The effect of conjugated linoleic acid on platelet function, platelet fatty acid composition, and blood coagulation in humans

Despite extensive research on conjugated linoleic acid (CLA) showing multiple beneficial effects in animal models, little is known about the role of dietary CLA in human health. To investigate if the beneficial effects of CLA seen in animal models are relevant to humans, we conducted a study with 17 healthy female volunteers who lived in the Metabolic Research Unit of the Western Human Nutrition Research Center for 93 d. This paper reports only the results from this study that are related to the effects of CLA supplementation on blood coagulation, platelet function, and platelet fatty acid composition. Throughout the study, the subjects were fed a low-fat diet (30 en% fat, 19 en% protein, and 51 en% carbohydrate) consisting 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) whose diet was supplemented with 3.9 g/d of CLA or a control group (n=7) who received an equivalent amount of sunflower oil consisting of 72.6% linoleic acid with no detectable CLA. Platelet aggregation was measured in platelet-rich plasma using adenosine diphosphate, collagen, and arachidonic acid agonists. No statistical difference was detected between the amount of agonist required to produce 50% aggregation of platelet-rich plasma before and after the subjects consumed the CLA, with the exception of a decrease in response to collagen. This decrease was found in both control and intervention groups with no significant difference between the groups, suggesting that both linoleic acid (sunflower oil) and CLA might have similar effects on platelet function. The prothrombin time, activated partial thromboplastin time, and the antithrombin III levels in the subjects were determined. Again, there was no statistically significant difference in these three parameters when pre-and post-CLA consumption values were compared. The in vivo bleeding times were also unaffected by CLA supplementation (10.4+2.8 min pre- and 10.2+1.6 min postconsumption). Platelet fatty acid composition was not markedly influenced by the consumption of dietary CLA, although there was a small increase in the amount of the 9 cis, 11 trans-18∶2 isomer normally present in platelets after feeding CLA for 63 days. In addition, small amounts of the 8 trans, 10 cis-18∶2 and the 10 trans, 12 cis-18∶2 isomers were detected in the platelets along with traces of some of the other isomers. Thus, when compared to sunflower     

Altered Lipid Response in Hamsters Fed <Emphasis Type="Italic">cis</Emphasis>-9,<Emphasis Type="Italic">trans</Emphasis>-11+<Emphasis Type="Italic">trans</Emphasis>-8,<Emphasis Type="Italic">cis</Emphasis>-10 Conjugated Linoleic Acid Mixture

The objective of the present study was to compare the effects of cis-9,trans-11 + trans-8,cis-10 conjugated linoleic acid (CLA) mixture to those of cis-9,trans-11 + trans-10,cis-12 CLA mixture and linoleic acid (LA) on lipoprotein profile, hepatic lipids, body composition and digestibility of dietary fat in hamsters (n = 17) fed diets containing 2% of experimental fat (w/w) for 28 days. The cis-9,trans-11 + trans-10,cis-12 CLA mixture showed higher LDL cholesterol concentrations than LA and the cis-9,trans-11 + trans-8,cis-10 CLA mixture. The cis-9,trans-11 + trans-8,cis-10 CLA mixture induced similar plasma LDL cholesterol and hepatic lipid concentrations, and coefficient of digestibility as LA, indicating no effect of the trans-8,cis-10 CLA isomer on these lipid parameters. On the other hand, the cis-9,trans-11 + trans-8,cis-10 CLA mixture induced higher plasma VLDL cholesterol and triglycerides than LA and the cis-9,trans-11 + trans-10,cis-12 CLA mixture. The cis-9,trans-11 + trans-8,cis-10 CLA mixture also induced the highest plasma glucose concentrations compared with the two other groups, indicating an impairment of glycemic control. No differences in body composition were noted between the three groups. The present results thus show that the cis-9,trans-11 + trans-8,cis-10 CLA mixture can deteriorate plasma VLDL cholesterol and triglycerides in hamsters, possibly due to an increased flux of glucose.     

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.     

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.     

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.     

Improved separation of conjugated fatty acid methyl esters by silver ion-high-performance liquid chromatography

Operating from one to six silver ion-high-performance liquid chromatography (Ag⁺-HPLC) columns in series progressively improved the resolution of the methyl esters of conjugated linoleic acid (CLA) isomeric mixtures from natural and commercial products. In natural products, the 8 trans, 10 cis-octadecadienoic (18∶2) acid was resolved from the more abundant 7 trans, 9 cis-18∶2, and the 10 trans, 12 cis-18∶2 was separated from the major 9 cis, 11 trans-18∶2 peak. In addition, both 11 trans, 13 cis-18∶2 and 11 cis, 13 trans-18∶2 isomers were found in natural products and were separated; the presence of the latter, 11 cis, 13 trans-18∶2, was established in commercial CLA preparations. Three Ag⁺-HPLC columns in series appeared to be the best compromise to obtain satisfactory resolution of most CLA isomers found in natural products. A single Ag⁺-HPLC column in series with one of several normal-phase columns did not improve the resolution of CLA isomers as compared to that of the former alone. The 20∶2 conjugated fatty acid isomers 11 cis, 13 trans-20∶2 and 12 trans, 14 cis-20∶2, which were synthesized by alkali isomerization from 11 cis, 14 cis-20∶2, eluted in the same region of the Ag⁺-HPLC chromatogram just before the corresponding geometric CLA isomers. Therefore, CLA isomers will require isolation based on chain length prior to Ag⁺-HPLC separation. The positions of conjugated double bonds in 20∶2 and 18∶2 isomers were established by gas chromatography-electron ionization mass spectrometry as their 4,4-dimethyloxazoline derivatives. The double-bond geometry was determined by gas chromatography-direct deposition-Fourier transform infrared spectroscopy and by the Ag⁺-HPLC relative elution order.     

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.     

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.     

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.     

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.     

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

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

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.     

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 conjugated linolenic acid and conjugated linoleic acid on lipid metabolism in mice

The effects of two isomers of conjugated linolenic acid (CLnA), α‐eleostearic acid (α‐ESA) and punicic acid (PA), on body fat and lipid metabolism were investigated, compared with a conjugated linoleic acid (CLA) mixture (primarily cis9,trans11‐ and trans10,cis12‐18:2) and α‐linolenic acid (ALA), a non‐conjugated octadecatrienoic acid, in the present study. ICR mice were fed either a control diet or one of four experimental diets supplemented with 1% α‐ESA, 1% PA, 1% CLA mixture and 1% ALA in the form of triacylglycerols (TAG) for 6 weeks. The weights of perirenal and epididymal adipose tissues were significantly decreased while the liver weight was significantly increased in mice fed CLA, compared with the control. In contrast to CLA, the tissue weights in α—ESA‐, PA‐ and ALA‐fed mice were not affected. No significant differences were observed in TAG, total cholesterol, high‐density lipoprotein and low‐density lipoprotein cholesterol levels among the five groups. The liver TAG level was significantly decreased in mice fed α‐ESA and PA while it was significantly increased in mice fed the CLA mixture. These results indicate that CLnA and CLA have differential effects on body fat mass and liver TAG levels in mice.     

Enzymatic enrichment of conjugated linoleic acid isomers and incorporation into triglycerides

A method was developed for the enrichment of either the cis9,trans11 or the trans10,cis12 isomer of conjugated linoleic acid (CLA) from a synthetic CLA mixture consisting predominantly of these isomers in equal amounts. Lipases were screened for their ability to selectively esterify one isomer at a significantly greater rate than the other isomer. An immobilized lipase from Rhizomucor miehei was nonselective, but a lipase from Geotrichum candidum esterified the cis9,trans11 isomer more rapidly than the trans10,cis12 isomer. This selectivity was exploited at the kilogram scale to prepare an ester fraction with a content of 91% cis9,trans11 CLA and an unreacted free fatty acid fraction consisting of 82% trans10,cis12 CLA, based on total CLA content. The components of the reaction mixture were separated by molecular distillation. Each enriched fraction was then incorporated into palm oil triglycerides by interesterification with the non-selective lipase from R. miehei. Two triglyceride fats resulted, which were enriched in either cis9,trans11 CLA (26.5% cis9,trans11 and 1.7% trans10,cis12) or trans10,cis12 CLA (3.5% cis9,trans11 and 22.9% trans10,cis12).     

Effect of pasture vs. concentrate diet on CLA isomer distribution in different tissue lipids of beef cattle

This study examined the effects of feeding pasture vs. concentrate on the distribution of CLA isomers in the lipids of longissimus and semitendinosus muscle, liver and heart muscle, and subcutaneous fat in beef bulls. Sixty-four German Holstein and German Simmental bulls were randomly allocated to either an indoor concentrate system or periods of pasture feeding followed by a finishing period on a concentrate containing linseed to enhance their beef content of n−3 PUFA and CLA. The concentrations of CLA isomers in the different tissues were determined by GC and silver ion HPLC. The diet affected the distribution of individual CLA isomers in the lipids of the different tissues. The concentration (mg/100 g fresh tissue) of the most prominent isomer, cis-9,trans-11 18∶2, was increased up to 1.5 times in liver and heart tissue of bulls fed on pasture as compared with concentrate. However, no diet effect was observed for cis-9,trans-11 18∶2 in the lipids of longissimus muscle and subcutaneous fat. In all tissues, the second-most abundant CLA isomer in concentratefed bulls was trans-7,cis-9 18∶2. In contrast, trans-11,cis-13 18∶2 was the second-most abundant CLA isomer in all investigated tissue lipids of pasture-fed bulls. The concentration of the trans-11,cis-13 18∶2 isomer was up to 15 times higher in tissues of pasture-fed bulls as compared with concentrate-fed animals. Furthermone, diet affected the concentrations of the CLA trans,trans 18∶2 isomers. Pasture feeding significantly increased the concentrations of some trans,trans 18∶2 isomers as compared with concentrate, predominantly trans-12,trans-14 18∶2 and trans-11,trans-13 18∶2. Overall, pasture feeding resulted in significantly increased concentrations of the sum of CLA isomers in the lipids of longissimus, muscle, subcutaneous fat, heart and liver muscle of German Holstein and German Simmental bulls, but not in semitendinosus muscle.     

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.     

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.     

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.     

A new conjugated linoleic acid isomer, 7 trans, 9 cis-octadecadienoic acid, in cow milk, cheese, beef and human milk and adipose tissue

The identity of a previously unrecognized conjugated linoleic acid (CLA) isomer, 7 trans, 9 cis-octadecadienoic acid (18∶2) was confirmed in milk, cheese, beef, human milk, and human adipose tissue. The 7 trans, 9 cis-18∶2 isomer was resolved chromatographically as the methyl ester by silver ion-high-performance liquid chromatography (Ag⁺-HPLC); it eluted after the major 9 cis, 11 trans-18∶2 isomer (rumenic acid) in the natural products analyzed. In the biological matrices in-vestigated by Ag⁺-HPLC, the 7 trans, 9 cis-18∶2 peak was generally due to the most abundant minor CLA isomer, ranging in concentration from 3 to 16% of total CLA. By gas chromatography (GC) with long polar capillary columns, the methyl ester of 7 trans, 9 cis-18∶2 was shown to elute near the leading edge of the major 9 cis, 11 trans-18∶2 peak, while the 4,4-dimethyloxazoline (DMOX) derivative permitted partial resolution of these two CLA isomers. The DMOX derivative of this new CLA isomer was analyzed by gas chromatography-electron ionization mass spectrometry (GC-EIMS). The double bond positions were at Δ7 and Δ9 as indicated by the characteristic mass spectral fragment ions at m/z 168, 180, 194, and 206, and their allylic cleavages at m/z 154 and 234. The cis/trans double-bond configuration was established by GC-direct deposition-Fourier transform infrared as evidenced from the doublet at 988 and 949 cm⁻¹ and absorptions at 3020 and 3002 cm⁻¹. The 7 trans, 9 cis-18∶2 configuration was established by GC-EIMS for the DMOX derivative of the natural products examined, and by comparison to a similar product obtained from treatment of a mixture of methyl 8-hydroxy-and 11-hydroxyoctadec-9 cis enoates with BF₃, in methanol. Contribution number S010 from the Food Research Center, Guelph, Ontario, Canada.