Inhibitory effect of conjugated linoleic acid on linoleic acid elongation in transformed yeast with human elongase

Conjugated linoleic acid (CLA; 18∶2) refers to a group of positional and geometric isomers derived from linoleic acid (LA; Δ9, 12–18∶2). Using a growing baker's yeast (Saccharomyces cerevisiae) transformed with human elongase gene, we examined the inhibitory effect of CLA at various concentrations (10, 25, 50, and 100 μM) on elongation of LA (25 μM) to eicosadienoic acid (EDA; Δ11,14–20∶2). Among four available individual CLA isomers, only c9,t11- and t10,c12-isomers inhibited elongation of LA to EDA. The extent of inhibition (ranging from 20 to 60%) was related to the concentration of CLA added to the medium. In the meantime, only these two isomers, when added at 50 μM to the media, were elongated to conjugated EDA (c11,t13- and t12,c14–20∶2) by the same recombinant elongase at the rate of 28 and 24%, respectively. The inhibitory effect of CLA on LA elongation is possibly due to competition between CLA isomers and LA for the recombinant elongase. Thus, results from this study and a previous study suggest that the biological effect of CLA is exerted through its inhibitory effect on Δ6-desaturation as well as elongation of LA which results in a decrease in long-chain n−6 fatty acids and consequently the eicosanoid synthesis.     

Conjugated linoleic acid production from linoleic acid by lactic acid bacteria

After screening 14 genera of lactic acid bacteria, Lactobacillus plantarum AKU 1009a was selected as a potential strain for CLA production from linoleic acid. Washed cells of L. plantarum with high levels of CLA production were obtained by cultivation in a nutrient medium with 0.06% (wt/vol) linoleic acid (cis-9,cis-12-octadecadienoic acid). Under the optimal reaction conditions with the free form of linoleic acid as the substrate, washed cells of L. plantarum produced 40 mg CLA/mL reaction mixture (33% molar yield) from 12% (wt/vol) linoleic acid in 108 h. The resulting CLA was a mixture of two CLA isomers, cis-9,trans-11 (or trans-9,cis-11)-octadecadienoic acid (CLA1, 38% of total CLA) and trans-9,trans-11-octadecadienoic acid (CLA2, 62% of total CLA), and accounted for 50% of the total FA obtained. A higher yield (80% molar yield to linoleic acid) was attained with 2.6% (wt/vol) linoleic acid as the substrate in 96 h, resulting in CLA production of 20 mg/mL reaction mixture [consisting of CLA1 (2%) and CLA2 (98%)] and accounting for 80% of total FA obtained. Most of the CLA produced was associated with the cells (ca. 380 mg CLA/g dry cells), mainly as FFA.     

Bioconversion of linoleic acid to conjugated linoleic acid by Lactobacillus reuteri under different growth conditions

BACKGROUND: Lactobacillus reuteri was grown in De Man/Rogosa/Sharpe (MRS) broth (initial pH 6.5) supplemented with free linoleic acid (LA) at different concentrations (5, 10, 20 and 30 mg mL^?1) and incubated aerobically at different temperatures (4, 10, 16, 22 and 30 °C) in order to test its ability to accomplish the bioconversion of LA to conjugated linoleic acid (CLA). Temperatures and LA concentrations producing the highest conversion of LA to CLA in the initial trials were tested further using micro‐anaerobic conditions and a lower initial pH (5.5). RESULTS: Data showed that production of CLA exhibited variations with regard to the fermentation conditions used. The highest production of CLA (0.108 mg mL^?1) was measured in a broth containing 20 mg mL^?1 free LA that was incubated aerobically at 10 °C for 30 h. When the initial pH of the reaction medium was reduced from 6.5 to 5.5, CLA production decreased. Micro‐aerobic conditions reduced the ability of Lb. reuteri to produce CLA, since production of CLA under aerobic conditions was at least 1.4 times greater. CONCLUSION: Production of CLA by Lb. reuteri at low temperatures and relatively high substrate concentrations provides novel opportunities for the development of functional foods with the benefits of enrichment in CLA and probiotic bacteria. Copyright © 2008 Society of Chemical Industry     

Conjugated linoleic acid suppresses triglyceride accumulation and induces apoptosis in 3T3-L1 preadipocytes

Four sets of experiments were conducted to examine the influence of conjugated linoleic acid (CLA) isomers during proliferation and differentiation of cultures of 3T3-L1 preadipocytes using physiological culturing conditions. Cultures treated with either albumin [bovine serum albumin (BSA) vehicle] or linoleic acid (LA) served as controls. For the proliferation study (Expt. 1), cells were cultured in media containing a crude mixture of CLA isomers or pure LA at 0, 10, 50, or 200 μM for 4 d. Preadipocyte proliferation (cell number, ³H-thymidine incorporation into DNA) decreased as the level of CLA increased in the cultures. In contrast, LA had no impact on DNA synthesis. In Experiment 2a, postconfluent cultures were grown in media containing a crude mixture of CLA isomers or LA at 0, 10, 50, or 200 μM for the next 6 d. Postconfluent cultures supplemented with 50–200 μM CLA had less triglyceride (TG) and were smaller in size than cultures supplemented with similar amounts of LA. In Experiment 2b, postconfluent cultures supplemented with 200 μM of a crude mixture of CLA isomers or LA were harvested on days 1, 3, 6, or 9. Differences in TG content of cultures supplemented with 200 μM CLA compared to control and LA-supplemented cultures became apparent after 3 d of culture. Experiments 3a and 3b examined whether the fatty acid vehicle (BSA vs. ethanol) or the vitamin E status (±0.2 mM α-tocopherol) of the cultures altered CLA’s impact on preadipocyte TG content. In Experiment 3a, ethanol-treated cultures had more TG than non-ethanol-treated cultures regardless of the fatty acid treatment. In Experiment 3b, cultures treated with 100 μM of either a crude mixture of CLA or the trans-10, cis-12 CLA isomer without supplemental vitamin E for 6 d had less TG than CLA-treated cultures containing vitamin E. In Experiment 4, postconfluent cultures were grown in media containing 100 μM LA or either a crude mixture of CLA isomers or the trans-10, cis-12 CLA isomer for 24–96 h to assess CLA’s influence on the cell cycle and indices of apoptosis. Cultures treated with 100 μM CLA for 24–96 h had more apoptotic cells than BSA- or LA-treated cultures. Furthermore, cultures treated for 48 h with CLA had fewer cells in the S-phase than control cultures. The effects of the trans-10,cis-12 CLA isomer were more pronounced than those of the crude mixture of CLA isomers. These data suggest that CLA may exert its antiobesity effects by inhibiting proliferation, attenuating TG content, and/or inducing apoptosis in (pre)adipocytes.     

In vitro genotoxicity/antigenotoxicity testing of some conjugated linoleic acid isomers using comet assay

Recently CLA isomers have received considerable attention as potential anti‐cancer agents. The aim of the study was to assess the genotoxicity/antigenotoxicity in vitro of linoleic acid (LA, c,c‐C18:2, Δ‐9), CLA isomer mixtures and homogeneous CLA TAGs (TriCLA) using the comet assay, to evaluate the effects on the extent of DNA injury in human hepatoma (HepG2) cells. The study was carried out both on commercial CLA (CLAc) and on CLA synthesized from grapestone oil (CLAg). The CLA isomer mixtures had different isomer profiles, determined by silver‐ion HPLC (Ag⁺‐HPLC), in particular CLAc was characterized by four main isomers (t8,c10; c9,t11; t10,c12; c11,t13), while CLAg showed two main isomers (c9,t11; t10,c12). As regards antigenotoxicity testing, LA, TriCLAg, and above all TriCLAc were effective antigenotoxic compounds against ethylmethanesulfonate (EMS) induced genotoxicity, while LA and CLAg were almost equally effective against 4‐nitroquinoline N‐oxide (4NQO) induced DNA damage. Both TriCLAc and TriCLAg showed an increased antigenotoxic activity toward EMS and a lower antigenotoxic activity toward 4NQO, with respect to both CLAc and CLAg. The higher capability of CLAg with respect to CLAc in counteracting the genotoxicity of 4NQO could be due to the different CLA isomer composition. Practical applications: CLA isomers have shown many beneficial health effects both on animals and humans. They are widely used in nutritional supplements, as CLA improves body composition by reducing fat storage. In this regard it is very important to know, besides the chemical and analytical aspects, also genotoxic and antigenotoxic effects of different CLA mixtures. To our best knowledge, few results have been reported on CLA antigenotoxic properties by the comet assay, and no data could be retrieved in the literature for TriCLA antigenotoxicity testing. The obtained results are interesting in that they can increase the knowledge on particular fatty acids used in commercial supplements.     

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.     

Conjugated linoleic acid isomers reduce blood cholesterol levels but not aortic cholesterol accumulation in hypercholesterolemic hamsters

The aim of the present study was to characterize plasma lipids and lipoprotein cholesterol and glucose concentrations in hamsters fed either cis-9, trans-11 CLA (9c, 11t CLA); trans-10, cis-12 CLA (10t, 12c CLA); or linoleic acid (LA) on the accumulation of aortic cholesterol in hypercholesterolemic hamsters. One hundred male F₁B strain Syrian Golden Hamsters (Mesocricetus auratus) (BioBreeders Inc., Watertown, MA) approximately 9 wk of age were housed in individual stainless stel hanging cages at room temperature with a 12-h light/dark cycle. Hamsters were given food and water ad libitum. Following a 1-wk period of acclimation, the hamsters were fed a chow-based (nonpurified) hypercholesterolemic diet (HCD) contaning 10% coconut oil (92% saturated fat) and 0.1% cholesterol for 2 wk. After an overnight fast, the hamsters were bled and plasma cholesterol concentrations were measured. The hamsters were then divided into 4 groups of 25 based on similar mean plasma VLDL and LDL cholesterol (non HDL-C) concentrations. Group 1 remained on the HCD (control). Group 2 was fed the HCD plus 0.5% 9c, 11t CLA isomer. Group 3 was fed the HCD plus 0.5% 10t, 12c CLA isomer. Group 4 was fed the HCD plus 0.5% LA. Compared with the control, both CLA isomers and LA had significantly lower plasma total cholesterol and HDL cholesterol concentrations (P<0.001) after 12 but not 8 wk of treatment and were not significantly different from each other. Also, both CLA isomers had significantly lower plasma non HDL-C concentrations (P<0.01) compared with the control after 12 but not 8 wk of treatment and were not significantly different from each other or the LA-fed hamsters. Plasma TG concentrations were significantly higher (P<0.004) with the 10t, 12c CLA isomer compared with the other treatments at 8 but not at 12 wk of treatment. Plasma TG concentrations were also significantly lower (P<0.03) with the 9c, 11t CLA isomer compared with the control at 12 wk of treatment. Also, the 10t, 12c CLA isomer and LA had significantly higher plasma glucose concentrations compared with the control and 9c, 11t CLA isomer (P<0.008) at 12 wk of treatment whereas at 8 wk, only the LA treatment had significantly higher plasma glucose concentrations (P<0.001) compared with the 9c, 11t CLA isomer. Although liver weights were significantly higher in 10t, 12c CLA isomer-fed hamsters, liver total cholesterol, free cholesterol, cholesterol ester, and TG concentrations were significantly lower in these hamsters compared with hamsters fed the control, 9c, 11t CLA isomer, and LA diets (P<0.05). The 9c, 11t CLA isomer and LA diets tended to reduce cholesterol accumulation in the aortic arch, whereas the 10t, 12c CLA isomer diet tended to raise cholesterol accumulation compared with the control diet; however, neither was significant. In summary, no differences were observed between the CLA isomers for changes in plasma lipids or lipoprotein cholesterol concentrations. However, the 9c, 11t CLA isomer did appear to lower plasma TG and glucose concentrations compared with the 10t, 12c CLA isomer. Such differences may increase the risk of insulin resistance and type 2 diabetes in humans when the 10t, 12c CLA isomer is fed separately.     

Variations in isomer distribution in commercially available conjugated linoleic acid

Conjugated linoleic acid (CLA) has been reported to have anticarcinogenic and antiatherogenic properties, to repartition body fat, to build bone mass, to normalize glucose tolerance, and to reduce hyperglycemia and diabetes. CLA products are now commercially available, and there is considerable interest in studying CLA because of this range of reported beneficial effects. However, little is known about the composition of these preparations. Representative commercial CLA products in capsule or liquid (aqueous or oily) form were analyzed for their CLA content and isomer composition using gas chromatography (GC), silver ion-high performance liquid chromatography (Ag⁺-HPLC) and spectroscopic techniques. The content of CLA in the preparations varied widely. Based on the GC-internal standard technique, total CLA varied from 20 to 89% by total weight and 28 to 94% of total fat. One product contained no CLA. The isomer distributions were generally of two types: those with two major CLA positional isomers, and those with four major CLA positional isomers. All the CLA preparations in capsule form contained the four isomer mixture, while the liquid preparations contained from two to four CLA positional isomers.     

Silver-ion high-performance liquid chromatographic separation and identification of conjugated linoleic acid isomers

This is the first report of the application of silverion impregnated high-performance liquid chromatography (Ag⁺-HPLC) to the separation of complex mixtures of conjugated linolenic acid (CLA) isomers present in commercial CLA sources and foods and in biological specimens. This method showed a clear separation of CLA isomers into three groups related to their trans,trans, cis,trans or trans,cis, and cis,cis configuration of the conjugated double-bound system. In addition, this method separated within each geometrical isomeric group. Following Ag⁺-HPLC isolation, gas chromatography (GC)-electron impact mass spectrometry, and GC-direct deposition-Fourier transformed infrared spectroscopy were used to confirm the identity of two major positional isomers in the cis/trans region, i.e., Δ8,10- and Δ11,13-octadecadienoic acid, which had not been chromatographically resolved previously, Furthermore, the potential of this method was demonstrated by showing different Ag⁺-HPLC profiles exhibiting patterns of isomeric distributions for biological specimens from animals fed a diet containing a commerical CLA preparation, as well as for a commerical cheese product.     

Decreased production of inflammatory mediators in human osteoarthritic chondrocytes by conjugated linoleic acids

Osteoarthritic chondrocytes (OC) produce excessive prostaglandin E₂ (PGE₂) and nitric oxide (NO), which function as inflammation mediators in the pathogenesis of osteoarthritis (OA). This study examined the effect of CLA alone and in combination with other PUFA on the FA composition and the production of PGE₂ and NO in OC cultures isolated from OA patients. Human OC were grown in monolayer and treated with one of the following PUFA treatments: CLA, CLA+arachidonic acid (CLA/AA), CLA_EPA (CLA/EPA), linoleic acid (LA), LA+AA (LA/AA), LA+EPA (LA/EPA), and ethanol (as a vehicle control) at 10 and 20 μM for 6 d. Supplementation of PUFA at 10 μM for 6 d did not introduce any cytotoxic effects or morphological changes in OC, whereas 20 μM resulted in apoptosis. Cultures of OC treated with CLA, CLA/AA, and CLA/EPA had higher concentrations of CLA isomers, and these isomers were not detected in other treatments. Supplementation of CLA or LA alone to the OC led to a lower PGE₂ production compared to the control. Combination of CLA/EPA resulted in the lowest PGE₂ production in cultured OC. OC cultures treated with CLA were lower in NO production than the control, whereas the LA/AA treatment demonstrated the lowest NO production. The fact that CLA alone or in combination with other PUFA modulated PGE₂ and NO production in human OC cultures suggests that these 18∶2 isomers may have the potential to influence OA pathogenesis.     

Conjugated linoleic acid isomer effects in atherosclerosis: Growth and regression of lesions

Conjugated linoleic acid (CLA), a mixture of positional and geometric isomers of octadecadienoic acid, has been shown to inhibit experimentally induced atherosclerosis in rabbits and also to cause significant regression of pre-established atheromatous lesions in rabbits. The two major CLA isomers (cis9,trans11 and trans10,cis12), now available at 90% purity, have been tested individually for their anti-atherogenic or lesion regression potency. The two major isomers and the mixture were fed for 90 d to rabbits fed 0.2% cholesterol. Atherosclerosis was inhibited significantly by all three preparations. The two CLA isomers and the isomer mix were also fed (1.0%) as part of a cholesterol-free diet for 90 d to rabbits bearing atheromatous lesions produced by feeding an atherogenic diet. A fourth group was maintained on a cholesterol-free diet. On the CLA-free diet atherosclerosis was exacerbated by 35%. Reduction of severity of atheromatous lesions was observed to the same extent in all three CLA-fed groups. The average reduction of severity in the three CLA-fed groups was 26±2% compared with the first control (atherogenic diet) and 46±1% compared with the regression diet. Insofar as individual effects on atherosclerosis were concerned, there was no difference between the CLA mix and the cis9,trans11 and trans10,cis12 isomers. They inhibit atherogenesis by 50% when fed as a component of a semipurified diet containing 0.2% cholesterol; and when fed as part of a cholesterol-free diet, they reduce established lesions by 26%. Reduction of atheromata to the observed extent by dietar means alone is noteworthy.     

Modulation of prostaglandin H synthase activity by conjugated linoleic acid (CLA) and specific CLA isomers

Conjugated linoleic acid (CLA) has been shown to inhibit tumorigenesis in animal models and is cytostatic to numerous cell lines in vitro. However, the mechanism of action is unknown. In the current study, we determined the effects of CLA and specific isomers of CLA on the rate of oxygenation of arachidonic acid by prostaglandin H synthase (PGHS) in ram seminal vesicle microsomes. The enzyme was incubated with 0.1 to 100 μM CLA or specific isomers of CLA for 2 min prior to the addition of 44 to 176 μM arachidonate. The isomers tested were 9(E),11(E) CLA; 9(Z),11(E) CLA; 9(Z),11(Z) CLA, and 10(E),12(Z) CLA. For a positive inhibitor control, flurbiprofen was used at 0.75 to 2.50 μM. Enzyme activity was assessed by measuring the rate of oxygen consumption. Inclusion of CLA or specific isomers of CLA in the incubation mixtures inhibits PGHS. The efficacy differs for each isomer, with the 9(Z),11(E) CLA isomer being the most effective and the 9(Z),11(Z) CLA isomer being the least effective inhibitor among the four CLA isomers tested. The K _i values obtained by Dixon replots range from 18.7 μM for the most effective isomer, 9(Z),11(Z) CLA, to 105.3 μM for the least effective isomer, 9(Z),11(Z) CLA. The K _i value for flurbiprofen with ram seminal vesicle microsomes was 0.33 μM. As the concentration of arachidonate was increased, the CLA-dependent inhibition of PGHS decreased, suggesting competitive inhibition. The results of this study demonstrate the potential of CLA and specific isomers of CLA to modulate prostaglandin biosynthesis.     

Lipase-catalyzed fractionation of conjugated linoleic acid isomers

The abilities of lipases produced by the fungus Geotrichum candidum to selectively fractionate mixtures of conjugated linoleic acid (CLA) isomers during esterification of mixed CLA free fatty acids and during hydrolysis of mixed CLA methyl esters were examined. The enzymes were highly selective for cis-9,trans-11–18∶2. A commercial CLA methyl ester preparation, containing at least 12 species representing four positional CLA isomers, was incubated in aqueous solution with either a commercial G. candidum lipase preparation (Amano GC-4) or lipase produced from a cloned high-selectivity G. candidum lipase B gene. In both instances selective hydrolysis of the cis-9,trans-11–18∶2 methyl ester occurred, with negligible hydrolysis of other CLA isomers. The content of cis-9,trans-11–18∶2 in the resulting free fatty acid fraction was between 94 (lipase B reaction) and 77% (GC-4 reaction). The commercial CLA mixture contained only trace amounts of trans-9,cis-11–18∶2, and there was no evidence that this isomer was hydrolyzed by the enzyme. Analogous results were obtained with these enzymes in the esterification in organic solvent of a commercial preparation of CLA free fatty acids containing at least 12 CLA isomers. In this case, G. candidum lipase B generated a methyl ester fraction that contained >98% cis-9,trans-11–18∶2. Geotrichum candidum lipases B and GC-4 also demonstrated high selectivity in the esterification of CLA with ethanol, generating ethyl ester fractions containing 96 and 80%, respectively, of the cis-9,trans-11 isomer. In a second set of experiments, CLA synthesized from pure linoleic acid, composed essentially of two isomers, cis-9,trans-11 and trans-10,cis-12, was utilized. This was subjected to esterification with octanol in an aqueous reaction system using Amano GC-4 lipase as catalyst. The resulting ester fraction contained up to 97% of the cis-9,trans-11 isomer. After adjustment of the reaction conditions, a concentration of 85% trans-10,cis-12–18∶2 could be obtained in the unreacted free fatty acid fraction. These lipase-catalyzed reactions provide a means for the preparative-scale production of high-purity cis-9,trans-11–18∶2, and a corresponding CLA fraction depleted of this isomer.     

Preparation of conjugated linoleic acid from safflower oil

Synthetically prepared mixtures of conjugated linoleic acid (CLA) are widely used in animal and cell culture studies to investigate the potential effects of the Δ9c, 11t-18:2 isomer found in food products from ruminant animals. Alkali isomerization of linoleic acid is a common method used in the synthesis of a mixture of CLA isomers containing predominantly the Δ9c, 11t-18:2 and Δ10t, 12c-18:2 isomers. Some biological activity might also be mediated by the Δ10t, 12c-18:2 isomer. Currently few published methodologies exist describing procedures for the enrichment of these two isomers. A method is described herein to take advantage of an inexpensive oil, safflower oil, for use in synthesis of CLA and a procedure to enrich the Δ10t, 12c-18:2 isomer.     

Photochemical production of conjugated linoleic acid from soybean oil

Conjugated linoleic acid (CLA), an anticarcinogenic compound with numerous other health benefits, is present mainly in dairy and beef lipids. The main CLA isomer present in dairy and beef lipids is cis 9, trans 11 CLA at a 0.5% concentration. The typical minimum human dietary intake of CLA is 10 times less than the 3 g/d suggested requirement that has been extrapolated from animal and cell-line studies. The objectives of this study were to produce CLA isomers from soybean oil by photoisomerization of soybean oil linoleic acid and to study the oxidation status of the oil. Refined, bleached, and deodorized soybean oil with added iodine concentrations of 0, 0.1, 0.25, and 0.5% was exposed to a 100-W mercury lamp for 0 to 120 h. An SP-2560 fused-silica capillary GC column with FID was used to analyze the esterified CLA isomers in the photoisomerized oil. The CLA content of the individual isomers was optimized by response surface methodology. Attenuated total reflectance (ATR)-FTIR spectra in the 3400 to 3600 cm⁻¹ range and ¹H NMR spectra in the 8 to 12 ppm range of the photoisomerized soybean oil were obtained to follow hydroperoxide formation. The largest amount of cis 9, trans 11 CLA isomer in soybean oil was 0.6%, obtained with 0.25% iodine and 84 h of photoisomerization. Lipid hydroperoxide peaks in the ATR-FTIR spectra and aldehyde peaks in the ¹H NMR spectra were not observed in the photoisomerized soybean oil, and the spectra were similar to that of fresh soybean oil. This study shows that CLA isomers can be produced simply and inexpensively from soybean oil by photoisomerization.     

Analysis of conjugated linoleic acid isomers and content in french cheeses

Conjugated linoleic acid (CLA) occurs in food as a result of microbial enzymatic reactions, free radical-type oxidation, and heat treatment. CLA is found in animal products, such as meat and dairy products, especially in cheeses. The CLA composition of 12 different French cheeses was determined by a combination of different analytical methods: reversed-phase high-performance liquid chromatography (RP-HPLC), gas chromatography-mass spectrometry (GC-MS), GC-Fourier transform infrared (GC-FTIR), and silver nitrate thin-layer chromatography (AgNO₃-TLC). New isomers (Δ8,10- and Δ11,13-octadecadienoic acids with all possible cis and trans configurations) that co-eluted with previously identified isomers (Δ9c,11t-; Δ9t,11c-; Δ10c,12t-; Δ10t,12c-; Δ11c,13c-; Δ9c,11c-; Δ10c,12c-; Δ9t,11t-; Δ10t12t-octadecadienoic acids) were detected. Δ9c,11t-Octadecadienoic acid was the major CLA isomer in these cheeses. All isomers were present in each product, whatever the production process. However, CLA content in the cheeses varied from 5.3 to 15.80 mg/g of cheese fat, which depended primarily on the origin of the milk (season, geography) and somewhat on the production process.     

Evidence that the trans-10,cis-12 isomer of conjugated linoleic acid induces body composition changes in mice

We investigated the effects of conjugated linoleic acid (CLA) preparations, which were enriched for the cis-9,trans-11 CLA isomer or the trans-10,cis-12 CLA isomer, on body composition in mice. Body composition changes (reduced body fat, enhanced body water, enhanced body protein, and enhanced body ash) were associated with feeding the trans-10,cis-12 CLA isomer. In cultured 3T3-L1 adipocytes, the trans-10,cis-12 isomer reduced lipoprotein lipase activity, intracellular triacylglycerol and glycerol, and enhanced glycerol release into the medium. By contrast, the cis-9,trans-11 and trans-9,trans-11 CLA isomers did not affect these biochemical activities. We conclude that CLA-associated body composition change results from feeding the trans-10,cis-12 isomer.     

Synthesis,isolation, and GC analysis of all the 6,8-to 13,15-<Emphasis Type="Italic">cis/trans</Emphasis> conjugated linoleic acid isomers

Octadecadienoic acids with conjugated double bonds are often referred to as conjugated linoleic acid, or CLA. CLA is of considerable interest because of potentially beneficial effects reported from animal studies. Analysis of CLA is usually carried out by GC elution of FAME. If the presence of low-level isomers is of interest, a complementary technique such as silverion HPLC is also used. These analyses have been hindered by a lack of well-characterized commercially available reference materials. Described here are the synthesis and isolation of selected 6,8-through 13,15-positional CLA isomers, followed by isomerization of these CLA isomers with iodine to produce all the possible cis,cis,cis,trans,trans,cis, and trans,trans combinations. Also present are the GC retention times of the CLA FAME relative to γ-linolenic acid (6c,9c,12c-octadecatrienoic acid) FAME using a 100-m CP Sil-88 capillary column (Varian Inc., Lake Forest, CA). These data include all the CLA isomers that have been identified thus far in foods and dietary supplements and should greatly aid in the future analysis of CLA in these products.     

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.     

The effect of conjugated linoleic acid on the antioxidant enzyme defense system in rat hepatocytes

Short-term effects of physiological concentrations of conjugated linoleic acid (CLA) on membrane integrity, metabolic function, cellular lipid composition, lipid peroxidation, and antioxidant enzymes were examined using rat hepatocyte suspension cultures. Incubation with CLA (5–20 ppm) for 3 h decreased the ability of hepatocyte plasma membranes to exclude trypan blue by approximately 25%, and caused leakage of cytosolic lactate dehydrogenase (LDH) into the medium. The significant decrease (P<0.02) in hepatocyte viability as measured by LDH leakage during cell incubation with 10 and 20 ppm CLA was not associated with significant changes in cellular ATP content. Protein synthesis in hepatocytes was elevated (P<0.05) in the presence of 5 and 10 ppm CLA, but at a higher concentration (20 ppm), protein synthesis was similar to that of control cells. Gluconeogenesis was maintained in cells incubated with lower concentrations of CLA (5 and 10 ppm) but was decreased (P<0.02) at the higher concentration. Incubation with 20 ppm CLA for 3 h did not affect the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme of cholesterol synthesis. Both cis-9,trans-11/trans-9,cis-11, and cis-10,trans-12/trans-10,cis-12 isomers of CLA were incorporated to a similar level into hepatocytes. Levels ranged from 3.9 to 4.1%, respectively, of total fatty acids in neutral lipids, and from 0.7 to 0.8%, respectively, of total fatty acids in phospholipids. Cellular lipid peroxidation remained unchanged in the presence of CLA (5–20 ppm), despite significant inhibition (P<0.05) of superoxide dismutase. Catalase activity was maintained near control levels in the presence of 5 and 10 ppm CLA but was significantly decreased in the presence of 20 ppm CLA. Glutathione peroxidase activity was significantly decreased in the presence of 10 ppm CLA. The apparent sensitivity of the antioxidant enzyme defense system of liver cells to CLA, coupled with the lack of effect of CLA on lipid peroxidation in cells, suggests that cytotoxic effects of CLA as described by LDH leakage and decreased gluconeogenesis were not mediated by a prooxidant action in hepatocytes.