Countercurrent approach to the enrichment of Δ9c,11t-and Δ10t,12c-18:2 isomers by urea complexation

CLA refers to a group of geometrical and positional isomers of linoleic acid. CLA has been shown to have potentially beneficial effects on cancer, atherosclerosis, and body metabolism in animals. Mixtures containing equal amounts of these isomers are commonly used in many research studies because of their greater availability and lower cost relative to pure isomers. This has hindered progress in elucidating the biological properties of specific isomers and their relevance in animal and human biology. A method was developed that offers a compromise between cost and utility to make available enriched mixtures of either the Δ9c,11t- or Δ10t,12c-18:2 isomers for use in a wide range of experimental applications. A countercurrent approach was developed to separate the Δ9c,11t- and Δ10t,12c-18:2 isomers from an equal mixture of these two isomers by urea complexation. After three successive rounds of complexation using an equal amount of CLA and urea, a fraction enriched in Δ9c,11t-18:2 containing 42.5 and 17.4% of Δ9c,11t-and Δ10t,12c-18:2, respectively, was recovered. After a single round of complexation using 2.5 g urea/g CLA, a fraction enriched in Δ10t,12c-18:2 was recovered containing 29.7 and 69.1% of Δ9c,11t- and Δ10t,12c-18:2, respectively.     

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.     

Changes in conjugated linolenic acid composition within samples obtained from a single source

Conjugated linoleic acid (CLA; 9c, 11t-18∶2) and CLA isomers have been reported, in animals, to exhibit a variety of health-related benefits. Silver ion high-performance liquid chromatography (Ag-HPLC) was found to provide better resolution of the isomes than gas chromatography. Most commercially available samples of CLA, prepared by base-catalyzed isomerization of linoleic acid (9c, 12c-18∶2), are conposed of mixtures of four major isomers. While these isomers have been characterized, we found significant changes in CLA isomer ratios within samples obtained from the same producer/commercial supplier over a period of 1.5 yr. In the first sample, the four cis/trans isomers (8t, 10c-18∶2, 9c, 11t-18∶2, 10t, 12c-18∶2 and 11c, 13t-18∶2) were present in a ratio of approximately 1∶2∶2∶1, while in the second sample they were present in almost equal proportions. If indeed certain daily levels of CLA intake are required to produce suggested health benefits in humans, changes in concentrations of specific CLA isomers could significantly impact these effects. Care must be taken to analyze the CLA used in human and animal studies.     

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.     

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.     

β-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.     

Dietary CLA Combined with Palm Oil or Ovine Fat Differentially Influences Fatty Acid Deposition in Tissues of Obese Zucker Rats

The effect of dietary conjugated linoleic acid (CLA) supplementation in combination with fat from vegetable versus animal origin on the fatty acid deposition, including that of individual 18:1 and 18:2 (conjugated and non-conjugated) isomers, in the liver and muscle of obese rats was investigated. For this purpose, 32 male Zucker rats were randomly assigned to one of four diets containing palm oil or ovine fat, supplemented or not with 1% of 1:1 cis(c)9,trans(t)11 and t10,c12 CLA isomers mixture. Total fatty acid content decreased in the liver and muscle of CLA-fed rats. In the liver, CLA increased saturated fatty acids (SFA) in 11.9% and decreased monounsaturated fatty acids (MUFA) in 6.5%. n-3 Polyunsaturated fatty acids (PUFA) relative proportions were increased in 30.6% by CLA when supplemented to the ovine fat diet. In the muscle, CLA did not affect SFA but decreased MUFA and PUFA percentages. The estimation of Δ9-indices 16 and 18 suggested that CLA inhibited the stearoyl-CoA desaturase activity in the liver (a decrease of 13–38%), in particular when supplemented to the ovine fat diet. Concerning CLA supplementation, the t10,c12 isomer percentage was 60–80% higher in the muscle than in the liver. It is of relevance that rats fed ovine fat, containing bio-formed CLA, had more c9,t11 CLA isomer deposited in both tissues than rats fed palm oil plus synthetic CLA. These results highlight the importance to further clarify the biological effects of consuming foods naturally enriched in CLA, alternatively to CLA dietary supplementation.     

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 Δ6 desaturation of linoleic and α-linolenic acids and on the Δ9 desaturation of stearic acid was investigated in vitro, using rat liver microsomes. The Δ6 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 Δ6 desaturation of α-linolenic acid was slightly affected by the presence of CLA isomers. The sole isomer to induce an inhibitory effect on the Δ9 desaturation of stearic acid was 10trans,12cis-18∶2.     

Consumption of c9,t11-18:2 or t10,c12-18:2 enriched dietary supplements does not influence milk macronutrients in healthy, lactating women

Substantial research suggests that the t10,c12–18:2, but not the c9,t11–18:2, isomer of conjugated linoleic acid (CLA) reduces milk fat synthesis in lactating bovine and rodent species. Because fat is the major energy-yielding component in human milk, we were interested in whether this is true for women as well. Thus, the effects of c9,t11–18:2 and t10,c12–18:2 on milk fat were examined in breast-feeding women (n = 12) in a double-blind, placebo-controlled, crossover study with latin-square design. The study was divided into six periods: baseline (3 days), three intervention periods (5 days each), and two washout periods (9 days each). During each intervention period, women consumed 750 mg/day of a supplement containing predominantly c9,t11–18:2, t10,c12–18:2, or 18:1 (olive oil placebo). Milk was collected by complete breast expression on the final day of each period. Infant milk consumption was estimated by 24 h weighing on the penultimate day of each intervention and washout period, and maternal adiposity (% body fat) was determined at baseline using dual energy X-ray absorptiometry. Milk c9,t11–18:2 and t10,c12–18:2 concentrations were greater (P < 0.05) during the corresponding CLA treatment periods as compared to the placebo period, providing strong evidence of subject compliance. Both CLA isomers were transferred into milk fat at relatively high efficiency; average transfer efficiency was estimated to be 23.3%. Compared to the placebo treatment, milk fat content was not reduced during either CLA treatment. Data indicate that body fatness did not modify any putative effect of isomeric CLA consumption on milk fat concentration. The evidence from this study suggests that the sensitivity of lactating women’s mammary tissue to an anti-lipogenic effect of the t10,c12–18:2 isoform of CLA may be less than previously hypothesized.     

Effect of dietary conjugated linoleic acid (CLA) on metabolism of isotope-labeled oleic,linoleic, and CLA isomers in women

The purpose of this study was to investigate the effect of dietary CLA on accretion of 9c-18∶1, 9c, 12c-18∶2, 10t, 12c-18∶2, and 9c, 11t-18∶2 and conversion of these FA to their desaturated, elongated, and chain-shortened metabolites. The subjects were six healthy adult women who had consumed normal diets supplemented with 6 g/d of sunflower oil or 3.9 g/d of CLA for 63 d. A mixture of 10t, 12c-18∶2-d ₄, 9c, 11t-18∶2-d ₆, 9c-18∶1-d ₈, and 9c, 12c-18∶2-d ₂, as their ethyl esters, was fed to each subject, and nine blood samples were drawn over a 48-h period. The results show that dietary CLA supplementation had no effect on the metabolism of the deuterium-labeled FA. These metabolic results were consistent with the general lack of a CLA diet effect on a variety of physiological responses previously reported for these women. The ²H-CLA isomers were metabolically different. The relative percent differences between the accumulation of 9c, 11t-18∶2-d ₆ and 10t, 12c-18∶2-d ₄ in plasma lipid classes ranged from 9 to 73%. The largest differences were a fourfold higher incorporation of 10t, 12c-18∶2-d ₄ than 9c, 11t-18∶2-d ₆ in 1-acyl PC and a two- to threefold higher incorporation of 9c, 11t-18∶2-d ₆ than 10t, 12c-18∶2-d ₄ in cholesterol esters. Compared to 9c-18∶1-d ₈ and 9c, 12c-18∶2-d ₂, the 10t, 12c-18∶2-d ₄ and 9c, 11t-18∶2-d ₆ isomers were 20–25% less well absorbed. Relative to 9c-18∶1, incorporation of the CLA isomers into 2-acyl PC and cholesterol ester was 39–84% lower and incorporation of 10t, 12c-18∶2 was 50% higher in 1-acyl PC. This pattern of selective incorporation and discrimination is similar to the pattern generally observed for trans and cis 18∶1 positional isomers. Elongated and desaturated CLA metabolites were detected. The concentration of 6c, 10t, 12c-18∶3-d ₄ in plasma TG was equal to 6.8% of the 10t, 12c-18∶2-d ₄ present, and TG was the only lipid fraction that contained a CLA metabolite present at concentrations sufficient for reliable quantification. In conclusion, no effect of dietary CLA was observed, absorption of CLA was less than that of 9c-18∶1, CLA positional isomers were metabolically different, and conversion of CLA isomers to desaturated and elongated metabolites was low.     

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.     

Rumenic Acid Significantly Reduces Plasma Levels of LDL and Small Dense LDL Cholesterol in Hamsters Fed a Cholesterol- and Lipid-Enriched Semi-Purified Diet

Conjugated linoleic acids (CLAs) consist of a series of positional and geometrical isomers of linoleic acid. CLA have been reported to beneficially affect cardiovascular risk factors in animal models. In order to assess the role of individual CLA isomers on lipoprotein cholesterol concentration, 30 hamsters were fed for 12 weeks an hyperlipidic diet containing pure cis-9,trans-11 CLA (c9,t11) or pure trans-10, cis-12 CLA (t10,c12) isomers given alone or as a mixture. Plasma total cholesterol, LDL and HDL cholesterol concentrations were significantly lower in the c9,t11 CLA isomer fed hamsters relative to the Control group, with the most substantially effect on LDL cholesterol (−56%; P < 0.05). Plasma triacylglycerol concentrations did not differ significantly regarding those two groups. Plasma cholesterol parameters showed a tendency to decrease in the t10,c12 CLA isomer and CLA mixture fed hamsters compared with the Control group, but differences were not significant. For the first time, the atherogenic fraction of small dense LDL was investigated. Plasma small dense LDL cholesterol concentration was lower in the c9,t11 CLA relative to Control, while the t10,c12 and CLA mixture groups showed only a non significant tendency to decrease. Taken together, these data indicate that feeding rumenic acid (c9,t11 CLA) may beneficially affect lipoprotein profile in hamster fed a cholesterol- and lipid-enriched semi-purified diet, when t10,c12 CLA isomer or CLA mixture would be less active.     

Geometry of conjugated double bonds of CLA isomers in a commercial mixture and in their hepatic 20∶4 metabolites

Rats were fed a fat-free diet for 2 wk. After this period, while maintaining the animals on the same diet, the rats were given intragastrically 180 mg per day of a mixture of conjugated linoleic acids (CLA) as triacylglycerols. Gas chromatography-mass spectrometry (GC-MS) analyses of this mixture, as well as hydrazine reduction and GC-MS and GC-Fourier transform infrared analyses of the resulting monoenes, revealed the presence of two major isomers, the 9c, 11t-and the 10t, 12c-18∶2 accompanied by smaller amounts of the 8t, 10c and the 11c,13t−18∶2 isomers. Minor quantities of cis,cis and trans,trans conjugated isomers also were detected. The total fatty acid methyl esters from the liver lipids were fractionated by reversed-phase high-performance liquid chromatography, and the fraction containing the 20∶4 isomers was further fractionated by silver nitrate thin-layer chromatography. A band containing two 20∶4 conjugated isomers was submitted to hydrazine reduction and the resulting monoenes analyzed by GC-MS as dimethyl-oxazoline derivatives. The two conjugated isomers were tentatively identified as 5c,8c,11c,13t–20∶4 and 5c,8c,12t,14c−20∶4. These could be formed by desaturation and elongation of the 9c,11t-and 10t,12c−18∶2 present in the commerical CLA mixture.     

Protective action of CLA against oxidative inactivation of paraoxonase 1, an antioxidant enzyme

The effect of CLA on paraoxonase 1 (PON1), one of the antioxidant proteins associated with HDL, was investigated for its protective action against oxidative inactivation as well as its stabilization activity. When cis-9 (c9),trans-11 (t11)-CLA and t10,c12-CLA were examined for their protective activity against ascorbate/Cu²⁻-induced inactivation of PON1 in the presence of Ca²⁺, two CLA isomers exhibited a remarkable protection (E _max, 71–74%) in a concentration-dependent manner (50% effective concentration, 3–4 μM), characterized by a saturation pattern. Such a protective action was also reproduced with oleic acid, but not linoleic acid. Rather, linoleic acid antagonized the protective action of CLA isomers in a noncompetitive fashion. Additionally, the two CLA isomers also protected PON1 from oxidative inactivation by H₂O₂ or cumene hydroperoxide. The concentration-dependent protective action of CLA against various oxidative inactivation systems suggests that the protective action of CLA isomers may be mediated through their selective binding to a specific binding site in a PON1 molecule. Separately, the inactivation of PON1 by p-hydroxymercuribenzoate (PHMB), a modifier of the cysteine residue, was also prevented by CLA isomers, suggesting the possible existence of the cysteine residue in the binding site of CLA. The c9,t11-CLA isomer seems to be somewhat more effective than t10,c12-CLA in protecting against the inactivation of PON1 by either peroxides or PHMB, in contrast to the similar efficacy of these two CLA isomers in preventing ascorbate/Cu²⁺-induced inactivation of PON1. Separately, CLA isomers successfully stabilized PON1, but not linoleic acid. These data suggest that the two CLA isomers may play a beneficial role in protecting PON1 from oxidative inactivation as well as in its stabilization.     

<Emphasis Type="Italic">9-trans,11-trans</Emphasis>-CLA: Antiproliferative and proapoptotic effects on bovine endothelial cells

Endothelial cell function can be influenced by nutrition, especially dietary FA and antioxidants. One class of dietary FA that is found in meat and dairy products derived from ruminant animals is conjugated linoleic acids (CLA). We have examined the effects of several CLA isomers on endothelial cell proliferation. 9t,11t-CLA was the only isomer that inhibited bovine arotic endothelial cell (BAEC) [³H]methylthymidine incorporation (I₅₀=35 μM), and this antiproliferative effect was time-dependent. A small decrease (20%) in cell number was observed only at the highest concentration (60 μM) tested. The 9c,11t-, 9c,11c-, 10t 12c-, and 11c,13t-CLA isomers did not exhibit any antiproliferative effects over a 5–60 μM concentration range. α-Tocopherol and BHT decreased BAEC proliferation, but pretreatment of cells with either of these antioxidants substantially attenuated the antiproliferative effect of 9t,11t-CLA. No difference in lipid peroxidation, as measured by the thiobarbituric acid assay for malondialdehyde, was observed on treatment of endothelial cells with either 9t,11t- or 9c,11t-CLA. However, a 43% increase in caspase-3 activity was observed after incubating BAEC with 9t,11t-CLA, suggesting that the antiproliferative effect of this isomer is partially due to an apoptotic pathway. In contrast to the above results with normal endothelial cells, these five CLA isomers all inhibited proliferation of the human leukemic cell line THP-1, with the 9t,11t isomer again being the most (I₅₀=60 μM) effective. These results confirm that different CLA isomers have different inhibitory potencies on the proliferation of normal and leukemic cells.     

Identification of conjugated linoleic acid isomers in cheese by gas chromatography, silver ion high performance liquid chromatography and mass spectral reconstructed ion profiles. Comparison of chromatographic elution sequences

Commercial cheese products were analyzed for their composition and content of conjugated linoleic acid (CLA) isomers. The total lipids were extracted from cheese using petroleum ether/diethyl ether and methylated using NaOCH₃. The fatty acid methyl esters (FAME) were separated by gas chromatography (GC), using a 100-m polar capillary column, into nine minor peaks besides that of the major rumenic acid, 9c, 11t-octadecadienoic acid (18∶2), and were attributed to 19 CLA isomers. By using silver ion-high performance liquid chromatography (Ag⁺-HPLC), CLA isomers were resolved into seven trans, trans (5–9%), three cis/trans (10–13%), and five cis, cis (<1%) peaks, totaling 15, in addition to that of the 9c, 11t-18∶2 (78–84%). The FAME of total cheese lipids were fractionated by semipreparative Ag⁺-HPLC and converted to their 4,4-dimethyloxazoline derivatives after hydrolysis to free fatty acids. The geometrical configuration of the CLA isomers was confirmed by GC-direct deposition-Fourier transform infrared, and their double bond positions were established by GC-electron ionization mass spectrometry. Reconstructed mass spectral ion profiles of the m+2 allylic ion and the m+3 ion (where m is the position of the second double bond in the parent conjugated fatty acid) were used to identify the minor CLA isomers in cheese. Cheese contained 7 t,9c-18∶2 and the previously unreported 11t, 13c-18∶2 and 12c, 14t-18∶2, and their trans,trans and cis,cis geometric isomers. Minor amounts of 8,10-, and 10, 12–18∶2 were also found. The predicted elution orders of the different CLA isomers on long polar capillary GC and Ag^*-HPLC columns are also presented.     

Effect of <Emphasis Type="Italic">trans</Emphasis>8, <Emphasis Type="Italic">cis</Emphasis>10+<Emphasis Type="Italic">cis</Emphasis>9, <Emphasis Type="Italic">trans</Emphasis>11 Conjugated Linoleic Acid Mixture on Lipid Metabolism in 3T3-L1 Cells

Evidence suggests that minor isomers of conjugated linoleic acid (CLA), such as trans8, cis10 CLA, can elicit unique biological effects of their own. In order to determine the effect of a mixture of t8, c10+c9, t11 CLA isomers on selected aspects of lipid metabolism, 3T3-L1 preadipocytes were differentiated for 8 days in the presence of 100 μM linoleic acid (LA); t8, c10+c9, t11 CLA; t10, c12+c9, t11 CLA or purified c9, t11 CLA. Whereas supplementation with c9, t11 and t10, c12+c9, t11 CLA resulted in cellular triglyceride (TG) concentrations of 3.4 ± 0.26 and 1.3 ± 0.11 μg TG/μg protein, respectively (P < 0.05), TG accumulation following treatment with CLA mixture t8, c10+c9, t11 was significantly intermediate (2.5 ± 0.22 μg TG/μg protein, P < 0.05) between the two other CLA treatments. However, these effects were not attributable to an alteration of the Δ⁹ desaturation index. Adiponectin content of adipocytes treated with t8, c10+c9, t11 mixture was similar to the individual isomer c9, t11 CLA, and both the t8, c10+c9, t11 and c9, t11 CLA groups were greater (P < 0.05) than in the t10, c12+c9, t11 CLA group. Overall, these results suggest that t8, c10+c9, t11 CLA mixture affects TG accumulation in 3T3-L1 cells differently from the c9, t11 and t10, c12 isomers. Furthermore, the reductions in TG accumulation occur without adversely affecting the adiponectin content of these cells.     

Occurrence of conjugated linoleic acid isomers in beef

The amounts of Δ9,Δ11-conjugated linoleic acid (CLA) isomers were determined in loin-associated fat samples of bulls (n=6) and steers (n=7) by capillary gas chromatography of fatty acid methyl ester (FAME) derivatives. The main CLA-isomer—18:2 c9,t11—provided approximately 0.76 ± 0.15% and 0.86 ± 0.15% of total FAME in bulls and steers, respectively. No differences (P>0.05) were observed between the CLA isomer distribution of bulls (t9,c11, 0.026 ± 0.014%; c9,c11, 0.015 ± 0.008%; and t9,t11, 0.029 ± 0.003%) and steers (t9,c11, 0.027 ± 0.014%; c9,c11, 0.015 ± 0.005%; and t9,t11, 0.030 ± 0.007%).     

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,11trans- 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∶1n−9, suggesting an alteration of the Δ9 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.     

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 Δ6-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 [1-¹⁴C]9c,11t–18∶2 was a poorer substrate for Δ6-desaturase than [1-¹⁴C]-9c,12c–18∶2. Next, in vitro elongation of 6c,9c,11t–18∶3 and 6c,9c,12c–18∶3 (γ-linolenic acid) was investigated in rat liver microsomes. Under elongation conditions, [1-¹⁴C]6c,9c,11t–18∶3 was 1.5-fold better converted into [3-¹⁴C]8c,11c,13t–20∶3 than [1-¹⁴C]6c,9c,12c–18∶3 into [3-¹⁴C]8c,11c,14c–20∶3. Finally, in vitro Δ5-desaturation of 8c,11c,13t–20∶3 compared to 8c,11c,14c–20∶3 was investigated. The conversion level of [1-¹⁴C]8c,11c,13t–20∶3 into [1-¹⁴C]5c,8c,11c,13t–20∶4 was 10 times lower than that of [1-¹⁴C]8c,11c,14c–20∶3 into [1-¹⁴C]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 Δ5-desaturase.