Trans-10,Cis-12 conjugated linoleic acid reduces triglyceride content while differentially affecting peroxisome proliferator activated receptor γ2 and aP2 expression in 3T3-L1 preadipocytes

A series of experiments was conducted using 3T3-L1 preadipocytes as the cell model to determine: (i) whether the triglyceride (TG)-lowering effects of a crude mixture of conjugated linoleic acid (CLA) isomers were due to a specific isomer of CLA and the timing of treatment, (ii) if CLA reduced TG content by inhibiting a key regulator of adipogenesis, (iii) if CLA incorporated into either neutral lipid or phospholipid cell fractions, and (iv) whether the effects of CLA treatment were reversible. Trans-10,cis-12 CLA reduced TG content, whereas the cis-9,trans-11 isomer increased TG content compared to vehicle [bovine serum albumin (BSA)] controls. Treatment with 50 μM trans-10,cis-12 CLA during the entire 6 d of differentiation reduced TG content to a greater extent than treatment during either the first 3 d or last 3 d of differentiation. Trans-10,cis-12 CLA treatment of preadipocyte cultures for 48 h increased peroxisome proliferator activated receptor γ2 (PPARγ2) protein expression compared to cultures treated with linoleic acid (LA) or the BSA controls. CLA had no effect on adipose P2 (aP2), a fatty acid-binding protein regulated by PPARγ2. Both the cis-9,trans-11 and the trans-10,cis-12 isomers of CLA were incorporated into neutral lipids and phospholipids. However, cis-9,trans-11 CLA levels were one- to twofold higher than trans-10,cis-12 CLA levels. Moreover, trans-10,cis-12 CLA treatment reduced cis-11 18∶1 concentrations in both neutral lipids and phospholipids while increasing cis-9 18∶1 and 18∶2 concentrations. Palmitoleic acid (16∶1) levels were also lower in the neutral lipid fraction of cultures treated with trans-10,cis-12 CLA. Supplementing trans-10,cis-12 CLA-treated cultures (50 μM) with increasing levels of LA resulted in a dose-dependent increase in TG content compared to cultures treated with 50 μM CLA alone. LA supplementation also prevented some of the morphological changes associated with trans-10,cis-12 CLA treatment as seen with scanning electron microscopy. Treatment with 50μM trans-10,cis-12 CLA for 6 d decreased PPARγ2 levels, and supplementation of CLA-treated cultures with LA increased PPARγ2 levels compared with cultures treated with CLA alone. Taken together, these data indicate that in cultures of 3T3-L1 preadipocytes: (i) trans-10,cis-12 CLA is the TG-lowering isomer of CLA, and its effects are dependent on dose, duration of treatment, and the amount of LA in the cultures; (ii) trans-10,cis-12 CLA treatment alters the monounsaturated fatty acid profile of neutral- and phospholipids of the cultures; and (iii) although acute (2−d) trans-10,cis-12 CLA treatment increased PPARγ2 protein levels, chronic (6−d) treatment decreased PPARγ2 levels.     

The effect of conjugated linoleic acid on plasma lipoproteins and tissue fatty acid composition in humans

Conjugated linoleic acid (CLA) has been suggested by some animal studies to possess antiatherogenic properties. To determine, in humans, the effect of dietary CLA on blood lipids, lipoproteins, and tissue fatty acid composition, we conducted a 93-d study with 17 healthy female volunteers at the Metabolic Research Unit of the Western Human Nutrition Research Center. Throughout the study, subjects were fed a low-fat diet [30 energy percent (en%) fat, 19 en% protein, and 51 en% carbohydrate] that consisted of natural foods with the recommended dietary allowances for all known nutrients. After a 30-d stabilization period, subjects were randomly assigned to either an intervention group (n=10) supplemented daily with capsules containing 3.9 g of CLA or a control group (n=7) that received an equivalent amount of sunflower oil. The CIA capsules (CLA 65%) contained four major cis/trans geometric isomers (11.4% 9 cis-,11 trans-18∶2; 10.8% 8 trans-,10 cis-18∶2; 15.3% 11 cis-,13 trans-18∶2; and 14.7% 10 trans-, 12 cis-18∶2) and their corresponding cis/cis (6.74% total) and trans/trans (5.99% total) varieties in smaller amounts. Fasting blood was drawn on study days 30 (end of the stabilization period), 60 (midpoint of the intervention period), and 93 (end of the intervention period). Adipose tissue samples were taken on days 30 and 93. CLA supplementation for 63 d did not change the levels of plasma cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, and triglycerides. The weight percentage of CLA in plasma increased from 0.28±0.06 to 1.09±0.31 (n=10, P<0.05) after the supplementation. The 9 cis-,11 trans-isomer was the most prominent variety followed by the 11 cis-,13 trans- and 10 trans-,12 cis-isomers in lesser amounts. CLA in adipose tissue was not influenced by the supplementation (0.79±0.18 to 0.83±0.19 wt%) (n=10) and the 9 cis-,11 trans-variety was the only isomer present. Thus, contrary to findings from some animal studies, CLA does not seem to offer health benefits, in the short term, regarding the prevention of atherosclerosis in humans. CLA supplementation for 2 mon did not alter the blood cholesterol or lipoprotein levels of healthy, normolipidemic subjects. The supplementation did increase CLA in the plasma but only 4.23% of the ingested CLA was present in the plasma at any given time. No adverse effect of CLA supplementation was detected in this study.     

In vitro comparison of hepatic metabolism of 9cis-11 trans and 10trans-12cis isomers of CLA in the rat

Hepatic metabolism of the two main isomers of CLA (9cis-11 trans, 10trans-12cisC_18∶2) was compared to that of oleic acid (representative of the main plasma FA) in 16 rats by using the in vitro method of incubated liver slices. Liver tissue samples were incubated at 37°C for 17h under an atmosphere of 95% O₂/5%CO₂ in a medium supplemented with 0.75 mM of FA mixture (representative of circulating nonesterified FA) and with 55 μM [1-¹⁴C]9cis-11 trans C_18∶2, [1-¹⁴C]10trans-12cis C_18∶2, or [1-¹⁴C]oleate. The uptake of CLA by hepatocytes was similar for both isomers (9%) and was three times higher (P<0.01) than for oleate (2.6%). The rate of CLA isomer oxidation was two times higher (49 and 40% of incorporated amounts of 9cis-11 trans and 10trans-12 cis, respectively) than that of oleate (P<0.01). Total oxidation of oleate and CLA isomers into [¹⁴CO₂] was low (2 to 7% of total oxidized FA) compared to the partial oxidation (93 to 98%) leading to the production of [¹⁴C] acid-soluble products. CLA isoemrs escaping from catabolism were both highly desaturated (26.7 and 26.8%) into conjugated 18∶3. Oleate and CLA isomers were mainly esterified into neutral lipids (30%). They were slowly secreted as parts of VLDL particles (<0.4% of FA incorporated into cells), the extent of secretion of oleate and of 10trans-12 cis being 2.2-fold higher than that of 9cis-11 trans (P<0.02). In conclusion, this study clearly showed that both CLA isomers were highly catabolized by hepatocytes, reducing their availability for peripheral tissues. Moreover, more than 25% of CLA escaping from catabolism was converted into conjugated 18∶3, the biological properties of which remain to be elucidated.     

Effects of specific conjugated linoleic acid isomers on growth characteristics in obese Zucker rats

Growing female obese Zucker (fa/fa) rats were treated (via intragastric gavage) for 21 d with either a (i) vehicle [corn oil; 0.9 g/kg body weight (BW)], (ii) CLA mixture [50∶50; trans-10,cis-12 and cis-9,trans-11 CLA], (iii) cis-9,trans-11 CLA, or (iv) trans-10,cis-12 CLA (CLA treatments at 1.5 g CLA/kg BW). Compared with controls, average daily gain (g/d) was reduced 24 and 44% by the CLA mixture and trans-10,cis-12 CLA, respectively There was no treatment effect on average whole-body (minus heart and liver) composition (dry matter basis): fat (70.2%), protein (21.0%), and ash (4.3%). Compared with animals treated with cis-9,trans-11 CLA, obese Zucker rats treated with trans-10,cis-12 and the CLA mixture had 7.8% more carcass water. Treatment had no effect on heart or liver weights or on heart or liver weights as a percentage of body weight, but compared with the other treatments trans-10,cis-12 CLA increased liver lipid contentby 33%. Hepatic lipid ratios of 16∶1/16∶0 and 18∶1/18∶0 (a proxy for Δ⁹-desaturase capability) were not affected by treatment (0.1 and 0.6, respectively). Simlar to previous reports, CLA increased hepatic lipid content and altered both liver and carcass FA composition (i.e., reduced arachidonic acid content), but the ability of CLA to manipulate body composition in obese Zucker rats remains questionable.     

<Emphasis Type="Italic">Trans</Emphasis>-7,<Emphasis Type="Italic">cis</Emphasis>-9 CLA is synthesized endogenously by Δ<Superscript>9</Superscript>-desaturase in dairy cowsin dairy cows

Cis-9,trans-11 and trans-7,cis-9 CLA are the most prevalent CLA isomers in milkfat. The majority of cis-9,trans-11 CLA is synthesized endogenously by Δ⁹-desaturase. We tested the hypothesis that trans-7,cis-9 CLA originates from endogenous synthesis by inhibiting Δ⁹-desaturase with a source of cyclopropene FA (sterculic oil: SO) or with a trans-10,cis-12 CLA supplement. Experiment 1 (four cows; Latin square) involved four treatments: control, SO, partially hydrogenated vegetable oil (PHVO), and PHVO+SO. Milk, plasma, and rumen fluid were collected. Experiment 2 treatments (four cows) were 0 or 14.0 g/d of 10,12 CLA supplement; milk and plasma were collected. Samples were analyzed by GC and Ag⁺-HPLC to determine FA. In Experiment 1, SO decreased milkfat content of trans-7,cis-9 CLA by 68 to 71% and cis-9,trans-11 CLA by 61 to 65%. In Experiment 2, the 10,12 CLA supplement decreased milkfat content of trans-7,cis-9 CLA and cis-9,trans-11 by 44 and 25%, respectively. Correcting for the extent of treatment-induced inhibition of Δ⁹-desaturase based on changes in myristic and myristoleic acids, endogenous synthesis of trans-7,cis-9 CLA represented 85 and 102% in Experiments 1 and 2, respectively. Similar corrected values were 77 and 58% for endogenous synthesis of cis-9,trans-11 CLA. Thus, milkfat cis-9,trans-11 CLA was primarily from endogenous synthesis with a minor portion from rumen escape. In contrast, trans-7,cis-9 CLA was not present in rumen fluid in significant amounts. Results indicate this isomer in milkfat is derived almost exclusively from endogenous synthesis via Δ⁹-desaturase.     

CLA isomers in milk fat from cows fed diets with high levels of unsaturated fatty acids

The concentrations of CLA isomers were determined by Ag⁺-HPLC in the milk fat of cows fed a control diet consisting of hay ad libitum and 15 kg of fodder beets or this diet supplemented with oilseeds containing either high levels of oleic acid (rapeseed), linoleic acid (sunflower seed), or α-linolenic acid (linseed). Highly significant (P≤0.001) correlations were found between the daily intakes of oleic acid and the concentration of the CLA isomer trans-7,cis-9 in milk fat; of linoleic acid and the CLA isomers trans-10,trans-12, trans-9,trans-11, trans-8,trans-10, trans-7,trans-9, trans-10,cis-12, cis-9,trans-11, trans-8,cis-10, and trans-7,cis-9; and of α-linolenic acid and the CLA isomers trans-12,trans-14, trans-11,trans-13, cis,trans/trans,cis-12,14, trans-11,cis-13, and cis-11,trans-13. CLA concentrations were also determined in the milk fat of cows grazing in the lowlands (600–650 m), the mountains (900–1210 m), and the highlands (1275–2120 m). The concentrations of many isomers were highest in milk fat from the highlands, but only three CLA isomers (cis-9,trans-11, trans-11,cis-13, and trans-8,cis-10) showed a nearly linear increase with elevation. Therefore, these three CLA isomers, and particularly the CLA isomer trans-11,cis-13, the second-most important CLA in milk fat from cows grazing at the three altitudes, could be useful indicators of milk products of Alpine origin.     

Effect of CLA on the cellular lipids of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The yeast Saccharomyces cerevisiae was cultivated in the presence of free CLA that was either a pure trans-10, cis-12 isomer, a pure cis-9, trans-11 isomer, or a 1∶1 mixture of the two, and the influence of these supplementations on the content and FA composition of the lipids in the yeast was determined. Neither the pure isomers nor their 1∶1 mixture influenced the growth of the yeast, but the trans-10, cis-12 isomer reduced the amount of cellular lipids by 40%. The reduction in total cellular lipids by the trans-10, cis-12 CLA was due to a reduction in TAG. Both of the isomers were incorporated into the yeast lipids, reaching a proportion of about 33% in TAG. With the incorporation of CLA, the yeast reduced the amount and desaturation of endogenously synthesized FA. These clear and pronounced isomer-specific effects of CLA on the yeast suggest that yeast might be a useful model to obtain a more comprehensive view of the mechanisms of the action of CLA on lipid metabolism.     

CLA production from ricinoleic acid by lactic acid bacteria

The ability to produce CLA from ricinoleic acid is widely distributed in lactic acid bacteria. Washed cells of Lactobacillus plantarum JCM 1551 were selected as a potential catalyst for CLA production from ricinoleic acid. Cells cultivated in medium supplemented with a mixture of α-linolenic acid and linoleic acid showed enhanced CLA productivity. Under optimal reaction conditions, with the free acid form of ricinoleic acid as the substrate and washed cells of L. plantarum as the catalyst, 2.4 mg/mL CLA was produced from 3.4 mg/mL ricinoleic acid in 90 h, with a molar yield with respect to ricinoleic acid of 71%. The CLA produced, which was obtained in the FFA form, consisted of a mixture of two CLA isomers, cis-9,trans-11-octadecadienoic acid (21% of total CLA) and trans-9,trans-11-octadecadienoic acid (79% of total CLA), and accounted for 72% of the total FA obtained.     

Effect of abomasal infusions of geometric isomers of 10,12 conjugated linoleic acid on milk fat synthesis in dairy cows

The trans-10, cis-12 isomer of conjugated linoleic acid (CLA) decreases TAG accumulation in 3T3-L1 adipocytes, reduces lipid accretion in growing animals, and inhibits milk fat synthesis in lactating mammals. However, there is evidence to suggest that other FA may also exert antilipogenic effects. In the current experiment, the effects of geometric isomers of 10,12 CLA on milk fat synthesis were examined using four Holstein-British Friesian cows in a 4×4 Latin Square experiment with 14-d periods. Treatments consisted of abomasal infusions of skim milk, or skim milk containing trans-10, cis-12 CLA (T1), trans-10, trans-12 CLA (T2), or a mixture of predominantly 10,12 isomers containing (g/100 g) trans-10, cis-12 (35.0), cis-10, trans-12 (23.2), trans-10, trans-12 (14.9), and cis-10, cis-12 (5.1). CLA supplements were prepared from purified ethyl linoleate and infused as nonesterified FA. Infusions were conducted over a 4-d period with a 10-d interval between treatments and targeted to deliver 4.5 g/d of 10,12 CLA isomers. Compared with the control, trans-10, trans-12 CLA had no effect (P>0.05) on milk fat yield, whereas treatments T1 and T3 depressed (P<0.05) milk fat content (19.8 and 22.9%, respectively) and decreased milk fat output (20.8 and 21.3%, respectively). Comparable reductions in milk fat synthesis to 4.14 and 1.80 g trans-10, cis-12/d supplied by treatments T1 and T3 indicate that other 10,12 geometric isomers of CLA have the potential to exert antilipogenic effects. The relative abundance of cis-10, trans-12 CLA in treatment T3 and the low transfer efficiency of this isomer into milk suggest that cis-10, trans-12 CLA was the active component.     

Differences in CLA isomer distribution of cow's milk lipids

The uniqueness of ruminant milk lipids is based on their high concentration of CLA. Maximal CLA concentrations in milk lipids require optimal conditions of ruminal fermentation and substrate availability, conditions like those present in pasture-fed cows. Our previous work showed that farm management (indoor feeding vs. pasture feeding) markedly influenced the CLA concentration. In this study, the objective was to evaluate the influence of the farm management system as dependent on different locations. Milk samples from different locations (Thuringia and the Alps, representing diverse altitudes) were collected during the summer months and analyzed for FA profile and CLA isomer distribution. The proportion of PUFA and total CLA in milk fat was significantly lower in milk from indoor cows compared with the pasture cows in the Alps. The trans-11 18∶1 in milk fat of Alpine cows was elevated, in contrast to lower values for trans-10 18∶1. Milk from cows grazing pasture in the Alps was higher in EPA and lower in arachidonic acid than milk from indoor-fed cows. The proportion of cis,trans/trans,cis isomers of CLA was 10 times higher from the indoor cows than from the Alpine cows. In addition to the major isomer cis-9,trans-11, this difference also occurred for the trans-11,cis-13 isomer, which represented more than a fourth of the total CLA present in milk fat. This is the first report showing a special isomer distribution in the milk fat of cows living under very natural conditions. We hypothesize that the CLA isomer trans-11,cis-13 is formed in large quantity as a result of grazing mountain pasture, which is rich in α-linolenic acid.     

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.     

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.     

Effects of the individual isomers cis-9,trans-11 vs. trans-10,cis-12 of conjugated linoleic acid (CLA) on inflammation parameters in moderately overweight subjects with LDL-phenotype B

Immune-modulating effects of CLA have been reported in animals, but results are inconsistent. In humans, CLA has shown no effects or only minor effects on immune function. The objective of this study was to evaluate the immune-modulating effects of 3 g cis-9,trans-11 (c9,t11) vs. trans-10,cis-12 (t10,c12) CLA isomers in a population with a high risk of coronary heart disease characterized by moderate overweight (body-mass index, 25–32.5 kg/m²) in combination with LDL-phenotype B (≥35% small LDL cholesterol, density≥1.040 g/mL). After a run-in period of 1 wk, 42 men and women were randomly allocated to the c9,t11 CLA group, the t10,c12 CLA group, or the placebo group. Effects of 13 wk of consumption of 3 g of CLA isomers on cytokine production by ex vivo lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMC) and whole blood, and on plasma C-remononuclear protein (CRP) concentrations were evaluated. To generate hypotheses for future studies, protein expression patterns of 42 cytokines, chemokines, and growth factors were evaluated with an antibody array in pooled, nonstimulated, fasting plasma samples. LPS induced interleukin (IL)-6, IL-8, and tumor necrosis factor-α production by PBMC, and whole blood as well as plasma CRP concentrations were not significantly changed by the c9,t11, and the t10,c12 CLA isomers. The cytokine expression profile in nonstimulated plasma suggested that both CLA isomers induced a specific inflammatory signature, in which the c9,t11 CLA group showed more activity in terms of numbers of proteins regulated. We conclude that daily consumption of 3 g of c9,t11 or t10,c12 CLA isomer did not affect LPS-stimulated cytokine production by PBMC or whole blood and plasma CRP levels. Inflammatory signatures in fasting, nonstimulated plasma as determined by an antibody array may indicate enhanced immune function by both CLA isomers.     

Changes in the milk and cheese fat composition of ewes fed commercial supplements containing linseed with special reference to the CLA content and isomer composition

A study was carried out to increase the CLA contents in ewes’ milk fat under field conditions by dietary means and to investigate the extent of the changes and consequences for milk processing and cheese quality. During a 3-mon period, ewes’ bulk milk samples were collected every week from two different herds. For the first 4 wk the ewes were fed a conventional diet. Then the following 6 wk a supplement enriched in α-linolenate (whole linseed) was incorporated into the ovine diet. Finally, in the last 3 wk the feeding was the same as in the first 4 wk. The FA profile in milk fat was monitored by GC, and the distribution of CLA isomers was thoroughly tested by combining GC-MS of 4,4-dimethyloxazoline derivatives (DMOX) with silver ion-HPLC (Ag⁺-HPLC) of FAME. Reconstructed mass spectral profiles of CLA characteristic ions from DMOX were used to identify positional isomers, and Ag⁺-HPLC was used to quantify them. An increase in total CLA in milk fat was observed, and total CLA remained elevated during the weeks of enriched α-linolenate feeding. In our experimental conditions there was a linear relationship between trans-vaccenic acid (trans-11-octadecenoic acid; trans-11 18∶1) and 9-cis, 11-trans CLA in ewes’ milk fat. Concerning the CLA isomer profile, increases in the 11,13- and 12,14–18∶2 positional isomers were considerable when linseed was included in the diet. Organoleptic characteristics of cheeses made with CLA-enriched milk did not substantially differ from those made with nonsupplemented ewes’ milk. CLA total content and isomer profile did not change during ripening.     

Effects of cis-9, trans-11 and trans-10, cis-12 CLA isomers on liver and adipose tissue fatty acid profile in hamsters

The aim of the present study was to investigate the effect of cis-9, trans-11 and trans-10, cis-12 CLA on FA composition of TAG in epididymal adipose tissue and liver, and of hepatic phospholipids PL. Twenty-four Syrian Golden hamsters were randomly divided into three groups of eight animals each and fed semipurified atherogenic diets supplemented with either 0.5 g/100g diet of linoleic acid or cis-9, trans-11 or trans-12, cis-9 CLA for 6 wk. Total lipids were extracted, and TAG and PL were separated by TLC. FA profile in lipid species from liver and adipose tissue, as well as in feces, was determined by GC. Trans-10, cis-12 CLA feeding significantly reduced linoleic and linolenic acids in TAG from both tissues, leading to reduced total PUFA content. Moreover, in the epididymal adipose tissue docosenoic and arachidonic acids were significantly increased. In liver PL, although no changes in individual FA were observed, total saturated FA (SFA) were decreased. No changes in TAG and PL FA profiles were induced by the cis-9, trans-11 CLA. TAG and PL incorporated cis-9, trans-11 more readily than trans-11, cis-12 CLA. This difference was not due to differential intestinal absorption, as shown by the analysis of feces. We concluded that only trans-10, cis-12 CLA induces changes in FA composition. Whereas increased PUFA content was observed in either liver or adipose tissue TAG, decreased SFA were found in liver PL. Incorporation of cis-9, trans-11 CLA in TAG is greater than that of trans-10, cis-12 CLA, but this is not due to differences in intestinal absorption.     

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.     

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.     

Milk fat synthesis is unaffected by abomasal infusion of the conjugated diene 18:3 isomers cis-6,trans-10, cis-12 and cis-6,trans-8,cis-12

It has been previously established that trans-10, cis-12 CLA is a potent inhibitor of milk fat synthesis. Although the mechanism of this action is not completely understood, it has been speculated that eicosanoid-like metabolites of this isomer formed by the activity of tissue desaturases may be responsible for its activity. The objective of this study was to investigate the effects of an enrichment containing an 18∶3 conjugated diene, produced in the metabolism of trans-10, cis-12 CLA, on milk fat synthesis. Three rumen-fistulated Holstein cows (210±8 d in milk) were randomly assigned in a 3×3 Latin square experiment. Treatments were (i) control, (ii) trans-10, cis-12 CLA supplement (2.1 g/d; positive control), (iii) enrichment providing two conjugated diene 18∶3 isomers (2.6 g/d of cis-6, trans-10, cis-12 and 4.0 g/d of cis-6, trans-8, cis-12) and trans-10, cis-12 CLA (2.1 g/d). Treatments were abomasally infused for 5 d at 4-h intervals, and there was a 7-d interval between periods. Milk yield, dry matter intake, and milk protein yield were unaffected by treatments. In contrast, the trans-10, cis-12 CLA supplement reduced milk fat yield by 27%, whereas the supplement enriched with conjugated diene 18∶3 isomers (treatment iii) had no effect on milk fat yield beyond that attributable to its trans-10, cis-12 CLA content. The transfer efficiency of trans-10, cis-12 CLA into milk fat was 25 and 24% for treatments ii and iii, respectively. At the same time, the abomasally infused conjugated diene 18∶3 isomers were transferred to milk fat with an efficiency of 33 and 41% for cis-6, trans-10, cis-12 and cis-6, trans-8, cis-12 18∶3, respectively. Overall, short-term abomasal infusion of the conjugated diene 18∶3 isomers had no effect on milk fat synthesis, thereby offering no support for an involvement of metabolies of trans-10, cis-12 CLA in the regulation of milk fat synthesis.     

Distributions of conjugated linoleic acid (CLA) isomers in tissue lipid classes of pigs fed a commercial CLA mixture determined by gas chromatography and silver ion-high-performance liquid chromatography

Pigs were fed a commercial conjugated linoleic acid (CLA) mixture, prepared by alkali isomerization of sunflower oil, at 2% of the basal diet, from 61.5 to 106 kg live weight, and were compared to pigs fed the same basal diet with 2% added sunflower oil. The total lipids from liver, heart, inner back fat, and omental fat of pigs fed the CLA diet were analyzed for the incorporation of CLA isomers into all the tissue lipid classes. A total of 10 lipid classes were isolated by three-directional thin-layer chromatography and analyzed by gas chromatography (GC) on long capillary columns and by silver-ion high-performance liquid chromatography (Ag⁺-HPLC); cholesterol was determined spectrophotometrically. Only trace amounts (<0.1%; by GC) of the 9,11–18∶2 cis/trans and trans, trans isomers were observed in pigs fed the control diet. Ten and twelve CLA isomers in the diet and in pig tissue lipids were sepatated by GC and Ag⁺-HPLC, respectively. The relative concentration of all the CLA isomers in the different lipid classes ranged from 1 to 6% of the total fatty acids. The four major cis/trans isomers (18.9% 11 cis, 13 trans-18∶2; 26.3% 10 trans, 12 cis-18∶2; 20.4% 9 cis, 11 trans-18∶2; and 16.1% 8 trans, 10 cis-18∶2) constituted 82% of the total CLA isomers in the dietary CLA mixture, and smaller amounts of the corresponding cis,cis (7.4%) and trans,trans (10.1%) isomers were present. The distribution of CLA isomers in inner back fat and in omental fat of the pigs was similar to that found in the diet. The liver triacylglycerols (TAG), free fatty acids (FFA), and cholesteryl esters showed a similar patterns to that found in the diet. The major liver phospholipids showed a marked increase of 9 cis,11 trans-18∶2, ranging from 36 to 54%, compared to that present in the diet. However, liver diphosphatidylglycerol (DPG) showed a high incorporation of the 11 cis,13 trans-18∶2 isomer (43%). All heart lipid classes, except TAG, showed a high content of 11 cis,13 trans-18∶2, which was in marked contrast to results in the liver. The relative proportion of 11 cis,13 trans-18∶2 ranged from 30% in the FFA to 77% in DPG. The second major isomer in all heart lipids was 9 cis,11 trans-18∶2. In both liver and heart lipids the relative proportions of both 10 trans,12 cis-18∶2 and 8 trans,10 cis-18∶2 were significantly lower compared to that found in the diet. The FFA in liver and heart showed the highest content of trans,trans isomers (31 to 36%) among all the lipid classes. The preferential accumulation of the 11 cis,13 trans-18∶2 into cardiac lipids, and in particular the major phospholipid in the inner mitochondrial membrane, DPG, in both heart and liver, appears unique and may be of concern. The levels of 11 cis,13 trans-18∶2 naturally found in foods have not been established.     

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.