Enrichment of CLA isomers by selective esterification with l-menthol using Candida rugosa lipase

Commercially available preparations of CLA are composed of almost equal amounts of 9-cis,11-trans (9c,11t)-CLA and 10-trans,12-cis (10t,12c)-CLA. Each isomer was fractionated and enriched, for availability as a food supplement, by a process comprising selective esterification with l-menthol by Candida rugosa lipase, distillation, and n-hexane extraction. The first selective esterification of CLA isomers was conducted with an equimolar amount of l-menthol of 30°C. The oil phase of the reaction mixture was fractionated into an l-menthyl ester fraction (9c,11t-CLA rich) and an FFA fraction (10t,12c-CLA rich) by distillation. The FFA fraction was esterified again with an equimolar amount of l-menthol to enrich 10t,12c-CLA. The 10t,12c-CLA preparation was obtained as the resulting FFA fraction by distillation. 10t,12c-CLA was enriched to 91% with 40% recovery. To enrich 9c,11t-CLA, the l-menthyl ester fraction in the first esterification was chemically hydrolyzed, and the resulting FFA were esterified again with an equimolar amount of l-menthol. The 9c, 11t-CLA preparation was obtained by chemical hydrolysis of the resulting l-methyl ester fraction, followed by n-hexane extraction. 9c,11t-CLA was enriched to 94% with 42% recovery. This effective process for purification of CLA isomers using l-methol is applicable to the production of food supplements.     

Selective Enrichment of Conjugated Linoleic Acid Isomers in Their Mixtures Using Combined Chemical and Enzymatic Methods

The aim of this study was to selectively enrich t10,c12-conjugated linoleic acid (t10,c12-CLA) and c9,t11-CLA in commercial CLA mixtures using a combination of urea crystallization and lipase-catalyzed esterification. The objective of the urea fractionation is to remove saturated and monounsaturated fatty acids (FA) from the CLA mixtures. CLA-enriched free FA (FFA) mixtures containing 53.8 wt% t10,c12-CLA and 39.1 wt% c9,t11-CLA were produced from the CLA mixtures containing ~34 wt% each of the two CLA isomers by a urea crystallization using methanol and the urea-to-FA weight ratio of 2.5:1. The CLA-enriched FFA mixtures were partially esterified with dodecan-1-ol in a recirculating packed-bed reactor using an immobilized lipase from Candida rugosa to further enrich the t10,c12-CLA and c9,t11-CLA in an FFA fraction and an FA dodecyl ester fraction, respectively, under the optimal conditions, i.e., temperature, 20 °C; FA-to-dodecan-1-ol molar ratio, 1:1; water content, 2 wt% of total substrates; residence time, 5 min; and reaction time, 24 h (for t10,c12-CLA enrichment) and 12 h (for c9,t11-CLA enrichment). After the reaction, an FFA fraction with 72.6 wt% t10,c12-CLA was obtained. Another FFA fraction with 62.0 wt% c9,t11-CLA was recovered after the saponification of the FA dodecyl ester fraction. The yields of t10,c12-CLA and c9,t11-CLA in the FFA fractions were 43.6 and 21.5 wt%, respectively, based on their initial weights in the CLA mixtures.     

Contrasting effects of t10,c12- and c9,t11-conjugated linoleic acid isomers on the fatty acid profiles of mouse liver lipids

The purpose of this study was to examine the effects of two purified isomers of CLA (c9,t11-CLA and t10,c12-CLA) on the weights and FA compositions of hepatic TG, phospholipids, cholesterol esters, and FFA. Eight-week-old female mice (n=6/group) were fed either a control diet or diets supplemented with 0.5% c9,t11-CLA or t10, c12-CLA isomers for 8 wk. Weights of liver total lipids and those of individual lipid fractions. did not differ between the control and the c9,t11-CLA groups. Livers from animals fed the t10,c12-CLA diet contained four times more lipids than those of the control group; this was mainly due to an increase in the TG fractions (fivefold), but cholesterol (threefold), cholesterol esters (threefold), and FFA (twofold) were also significantly increased. Although c9,t11-CLA did not significantly alter the weights of liver lipids when compared with the control group, its intake was associated with significant reductions in the weight percentage (wt% of total FAME) of 18∶1n−9 and 18∶1n−7 in the TG fraction and with significant increases in the weight percentage of 18∶2n−6 in the TG, cholesterol ester, and phospholipid fractions. on the other hand, t10,c12-CLA intake was linked with a significant increase in the weight percentage of 18∶1n−9 and a decrease in that of 18∶2n−6 in all lipid fractions. These changes may be the result of alterations in the activity of Δ9-desaturase (stearoyl CoA desaturase) and the enzymes involved in the metabolism of 18∶2n−6. Thus, the two isomers differed not only in their effects on the weights of total liver lipids and lipid fractions but also on the FA profile of the lipid fractions.     

cis-9,trans-11 and trans-10,cis-12 CLA affect lipid metabolism differently in primary white and brown adipocytes of Djungarian hamsters

We explored whether CLA isomers and other C₁₈ FA affect (i) lipid content and FA concentrations in total adipocyte lipids, (ii) FA synthesis from glucose in TAG and phospholipids of primary brown (BAT) and white adipocytes (WAT), and (iii) mRNA expression of uncoupling protein 1 (UCP1) in primary brown adipocytes of Djungarian hamsters (Phodopus sungorus). c9,t11-CLA, oleic, linoleic, and α-linolenic acid increased whereas t10,c12-CLA decreased lipid accumulation in both adipocyte types. t10,c12-CLA treatment affected FA composition mainly in BAT cells. CLA incorporation into lipids, in particular c9,t11-CLA, was higher in BAT. In both cell types, t10,c12-CLA treatment reduced the incorporation of glucose ¹³C carbon into FA of TAG and phospholipids, whereas c9,t11-CLA, linoleic, and α-linolenic acid either did not influence or dose-dependently increased glucose carbon incorporation into FA. UCP1 mRNA expression was inhibited by t10,c12-CLA but increased by c9,t11-CLA, linoleic, and α-linolenic acid. It is concluded that c9,t11-CLA and t10,c12-CLA have distinctly different effects on lipid metabolism in primary adipocytes. The effects of c9,t11-CLA are similar to those of other unsaturated C₁₈ FA. The opposite effects of c9,t11-CLA and t10,c12-CLA are evident in both WAT and BAT cultures; however, brown adipocytes seem to be more susceptible to CLA treatment.     

Analysis of CLA Isomer Distribution in Nutritional Supplements by Single Column Silver-Ion HPLC

Nutritional supplements containing conjugated linoleic acid (CLA) isomers, widely used to improve body composition and inhibit fat storage, should be thoroughly analyzed, as CLA effects are isomer specific. In this research, an improvement of silver-ion high performance liquid chromatography (Ag⁺-HPLC) was obtained by using a single-column instead of the most commonly used multi-columns. To develop the procedure, a standard CLA mixture has been derivatized with different chain length alcohols (from methanol to hexanol). Hexyl CLA showed better separation between CLA isomer pairs, in particular the resolution of the t10,c12: c9,t11 pair increased from 0.3 (methyl esters) to 0.8 (hexyl esters). Therefore, the CLA isomer composition of some commercial CLA products was determined by Ag⁺-HPLC analysis of hexyl esters. The main isomers in all supplements turned out to be c9,t11-CLA and t10,c12-CLA, the most anti-adipogenic isomer. t8,c10- and c11,t13-CLA were not detected. The nutritional supplements were also analyzed by high resolution gas chromatography of methyl esters to evaluate the fatty acid % composition and total CLA % content. The total CLA ranged from 79.4 to 94.4 %, and the t,t isomer from 1.4 to 7.3 %.     

Production of MAG of CLA by esterification with dehydration at ordinary temperature using <Emphasis Type="Italic">Penicillium camembertii</Emphasis> lipase

Production of MAG with CLA using Penicillium camembertii mono- and diacylglycerol lipase (referred to as lipase) was attempted for the purpose of expanding the application of CLA. The commercial product of CLA (referred to as FFA-CLA) is a FFA mixture containing almost equal amounts of 9cis,11trans (9c,11t)-CLA and 10t,12c-CLA. Esterification of FFA-CLA with glycerol without dehydration achieved 84% esterification but produced almost equal amounts of MAG and DAG. Esterification with dehydration not only achieved a high degree of esterification but also suppressed the formation of DAG. When a mixture of FFA-CLA/glycerol (1∶2, mol/mol), 1% water, and 200 units/g-mixture of P. camembertii lipase was agitated at 30°C for 72 h with dehydration at 5 mm Hg, the degree of esterification reached 95% and the contents of MAG and DAG were 90 and 6 wt%, respectively. This reaction system may be applied to the industrial production of MAG with unstable CLA.     

<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.     

Determination of <Emphasis Type="Italic">c</Emphasis>9,<Emphasis Type="Italic">t</Emphasis>11-CLA in major human plasma lipid classes using a combination of methylating methodologies

Isomeric CLA exhibit several significant biological activities in animals and humans and are easily isomerized to their corresponding t,t-CLA isomers during methylation with various acid-catalyzed reagents. To minimize such isomerization and provide a valid quantification of human plasma CLA content, several methylation methods were tested. Plasma neutral lipid, nonesterified FA (NEFA), and polar lipid classes were separated into the following fractions: (i) cholesteryl ester (CE, 1.2 mg/12 mL, 37.5% lipids), (ii) TAG (0.8 mg/12 mL, 25% lipids), (iii) NFFA (0.2 mg/12 mL, 6.2% lipids), (iv) MAG/DAG/cholesterol (0.3 mg/12 mL, 9.4% lipids), and (v) phospholipid (PL, 0.5 mg/20 mL, 15.6% lipids). Data showed that c9,t11-CLA found in TAG, MAG/DAG/cholesterol, and PL fractions were converted to methyl esters with sodium methoxide within 2 h at 55°C. However, the c9,t11-CLA in the CE fraction could not be completely converted to methyl esters by sodium methoxide/acetylchloride in methanol or methanolic KOH; instead, CE was treated with sodium methoxide and methyl acetate in diethyl ether for 1 h. NEFA were converted to methyl esters with trimethylsilyldiazomethane (TMSDAM). All reaction mixtures were monitored by TLC prior to GLC analysis. The highest enrichment of c9,t11-18∶2 (% FA) was in TAG (0.31%), followed by CE (0.14%) and PL (0.13%). The above methylation methods were then applied to a small subset (n=10) of nonfasting plasma lipid fractions to confirm the applicability of these data. Results from this subset of samples also indicated that the greatest enrichment of c9,t11-CLA was present in the TAG fraction (0.39%), followed by CE (0.27%) and PL (0.22%). These data indicate that different plasma fractions have different c9,t11-CLA contents.     

Preparation of regioisomers of structured TAG consisting of one mole of CLA and two moles of caprylic acid

TAG (MLM) with medium-chain FA (MCFA) at the 1,3-positions and long-chain FA (LCFA) at the 2-position, and TAG (LMM) with LCFA at the 1(3)-position and MCFA at 2,3(1)-positions are a pair of TAG regioisomers. Large-scale preparation of the two TAG regioisomers was attempted. A commercially available FFA mixture (FFA-CLA) containing 9-cis, 11-trans (9c, 11t)- and 10t,12c-CLA was selected as LCFA, and caprylic acid (C₈FA) was selected as MCFA. The MLM isomer was synthesized by acidolysis of acyglycerols (AG) containing two CLA isomers with C₈FA: A mixture of AG-CLA/C₈ FA (1∶10, mol/mol) and 4 wt% immobilized Rhizomucor miehei lipase was agitated at 30°C for 72 h. The ratio of MLM to total AG was 51.1 wt%. Meanwhile, LMM isomer was synthesized by acidolysis of tricaprylin with FFA-CLA: A mixture of tricaprylin/FFA-CLA (1∶2, mol/mol) and 4 wt% immobilized R. miehei lipase was agitated at 30°C for 24 h. The ratio of LMM to total AG was 51.8 wt%. MLM and LMM were purified from 1,968 and 813 g reaction mixtures by stepwise short-path distillation, respectively. Consequently, MLM was purified to 92.3% with 49.1% recovery, and LMM was purified to 93.2% with 52.3% recovery. Regiospecific analyses of MLM and LMM indicated that the 2-positions of MLM and LMM were 95.1 mol% LCFA and 98.3 mol% C₈ FA, respectively. The results showed that a process comprising lipase reaction and short-path distillation is effective for large-scale preparation of high-purity regiospecific TAG isomers.     

A novel method for solvent fractionation of two CLA isomers

Conjugated linoleic acid (CLA) is commercially available as a mixture consisting of almost equal amounts of the cis-9,trans-11-CLA (c9, t11) and trans-10, cis-12-CLA (t10, c12) isomers. Separation of the two isomers is highly significant since each exhibits different biochemical properties. Highly efficient separation could be accomplished by crystallization in acetone (solvent) of the two CLA isomers (solutes) in the presence of medium-chain fatty-acid (MCFA) additives. The relative concentration ratios of the two CLA isomers in the solvent-crystallized materials varied depending on which MCFA were added. Addition of lauric and decanoic acids resulted in the crystals predominantly containing t10,c12, whereas octanoic acid yielded those predominantly containing c9,t11. We have confirmed that onetime solvent crystallization using decanoic acid and octanoic acid additives increased the t10,c12 and c9,t11 concentrations, and that repeated solvent crystallization resulted in the ratio of c9,t11 to t10,c12 of at least 4∶96 or 98∶2.     

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.     

Enzymatic Synthesis of Diacylglycerols Enriched with Conjugated Linoleic Acid by a Novel Lipase from <Emphasis Type="Italic">Malassezia globosa</Emphasis>

Diacylglycerols (DAG) of conjugated linoleic acid (CLA) were prepared by esterification of glycerol with fatty acids enriched with CLA (FFA–CLA, >95%) in the presence of a novel lipase from Malassezia globosa (SMG1). Lipase SMG1 is strictly specific to mono- and diacylglycerols but not triacylglycerols, which is similar to the properties of lipase from Penicillium camembertii (lipase G 50), but lipase SMG1 showed preference on the production of DAG with the reaction proceeding. Low temperature was beneficial for the conversion of FFA–CLA into acylglycerols, the degree of esterification reached 93.0% when the temperature was 5 °C. The maximum DAG content (53.4%) was achieved at 25 °C. The rate of DAG synthesis increased as the enzyme loading increased. However, at lipase amounts above 240 U/g mixtures, no significant increases in DAG concentration were observed. The molar ratio of FFA–CLA to glycerol and initial water content were optimized to be 1:3 (mol/mol) and 3%. Lipase SMG1 showed no regioselectivity because the contents of 1,3-DAG and 1,2-DAG were 43.1% and 21.2% based on total content of acylglycerols. By calculating the ratio of 9c, 11t-CLA to 10t, 12c-CLA, it was indicated that lipase SMG1 showed a little preference to 10t, 12c-CLA at the sn-1(3) position of monoacylglycerols (MAG), while no selectivity for 9c, 11t-CLA at the sn-2 position of DAG was obviously found.     

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.     

Oxidation rate of conjugated linoleic acid and conjugated linolenic acid is slowed by triacylglycerol esterification and α-tocopherol

We have recently shown that α-eleostearic acid (α-ESA), a conjugated linolenic acid, has a stronger antitumor effect than conjugated linoleic acid (CLA), both in vitro and in vivo. In this study, the oxidative stability of α-ESA was examined compared with linoleic acid (LA), α-linolenic acid (LnA), and CLA. Thin layers of the FA (LA, 9Z, 11E-CLA, 10E,12Z-CLA, LnA, and α-ESA) were auto-oxidized at 37°C, and the FA remaining, the absorbed oxygen volume, the lipid hydroperoxide content, and the TBARS content were determined. The oxidation rate of α-ESA was faster than that of the unconjugated FA and CLA (9Z,11E-CLA and 10E,12Z-CLA). However, the lipid hydroperoxide and TBARS contents following α-ESA oxidation were low, suggesting production of only small amounts of rapid-reacting secondary oxidation products. Furthermore, the oxidative stability of conjugated FA (CLA and CLnA) in which the carboxylic acid group was esterified with triacylglycerol was greater than that of the FFA. Addition of an antioxidant (α-tocopherol) also increased the stability of the conjugated FA to a level similar to that of the unconjugated FA.     

Oxidation kinetics for <Emphasis Type="Italic">cis</Emphasis>-9,<Emphasis Type="Italic">trans</Emphasis>-11 and <Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12 isomers of CLA

The autoxidation processes of the cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12) isomers of CLA were separately observed at ca. 0% RH and different temperatures. The t10,c12 CLA oxidized faster than the c9,t11 isomer at all tested temperatures. The first half of the oxidation process of t10,c12 CLA obeyed an autocatalytic-type rate expression, but the latter half followed first-order kinetics. On the other hand, the entire oxidation process of c9,t11 CLA could be expressed by the autocatalytic-type rate expression. The apparent activation energies and frequency factors for the autoxidation of the isomers were estimated from the rate constants obtained at various temperatures based on the Arrhenius equation. The apparent activation energies for the CLA isomers were greater than those for the nonconjugated n−6 and n−3 PUFA or their esters. However, the enthalpyentropy compensation held during the autoxidation of both the CLA and PUFA. This suggested that the autoxidation mechanisms for the CLA and PUFA were essentially the same.     

Modulation of body composition and immune cell functions by conjugated linoleic acid in humans and animal models: Benefits vs. risks

We have reviewed the published literature regarding the effects of CLA on body composition and immune cell functions in humans and in animal models. Results from studies in mice, hamsters, rats, and pigs generally support the notion that CLA reduced depot fat in the normal or lean strains. However, in obese rats, it increased body fat or decreased it less than in the corresponding lean controls. These studies also indicate that t10,c12-CLA was the isomer that reduced adipose fat; however, it also increased the fat content of several other tissues and increased circulating insulin and the saturated FA content of adipose tissue and muscle. Four of the eight published human studies found small but significant reductions in body fat with CLA supplementation; however, the reductions were smaller than the prediction errors for the methods used. The other four human studies found no change in body fat with CLA supplementation. These studies also report that CLA supplementation increased the risk factors for diabetes and cardiovascular disease including increased blood glucose, insulin, insulin resistance, VLDL, C-reactive protein, lipid peroxidation, and decreased HDL. Most studies regarding the effects of CLA on immune cell functions have been conducted with a mixture of isomers, and the results have been variable. One study conducted in mice with the purified c9,t11-CLA and t10,c12-CLA isomers indicated that the two isomers have similar effects on immune cell functions. Some of the reasons for the discrepancies between the effects of CLA in published reports are discussed. Although significant benefit to humans from CLA supplementation is questionable, it may create several health risks in both humans and animals. On the basis of the published data, CLA supplementation of adult human diets to improve body composition or enhance immune functions cannot be recommended at this time.     

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.     

Maternal dietary alpine butter intake affects human milk: Fatty acids and conjugated linoleic acid isomers

Consumption of CLA by lactating women affects the composition of their milk, but the pattern of the different CLA isomers is still unknown. We determined the effects of short maternal supplementation with CLA-rich Alpine butter on the occurrence of FA and CLA isomers in human milk. In an open randomized controlled study with a two-period cross-over design, milk FA and CLA isomer concentrations were measured on postpartum days >-20 in two parallel groups of lactating women before, during, and after consumption of defined quantities of Alpine butter or margarine with comparable fat content (10 d of butter followed by 10 d of margarine for one group, and vice versa in the other). In the 16 women who completed the study (8/group), Alpine butter supplementation, increased the C₁₆ and C₁₈ FA, the sum of saturated FA, the 18∶1 trans FA, and the trans FA with CLA. The CLA isomer 18∶2 c9, t11 increased by 19.7%. Significant increases were also found for the isomers t9,t111, t7,c9,t11,c13, and t8,c10 18∶2. The remaining nine of the total 14 detectable isomers showed no changes, and concentrations were <5 mg/100g fat. A breastfeeding mother can therefore modulate the FA/CLA supply of her child by consuming Alpine butter. Further studies will show whether human milk containing this FA and CLA isomer pattern acts as a functional food for newborns.     

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.     

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.