Differential Inflammatory Responses in Cultured Endothelial Cells Exposed to Two Conjugated Linoleic Acids (CLAs) under a Pro-Inflammatory Condition

Conjugated linoleic acid (CLA) isomers have been shown to possess anti-atherosclerotic properties, which may be related to the downregulation of inflammatory pathways in different cell types, including endothelial cells (ECs). However, whether different CLA isomers have different actions is not entirely clear, with inconsistent reports to date. Furthermore, in cell culture studies, CLAs have often been used at fairly high concentrations. Whether lower concentrations of CLAs are able to affect EC responses is not clear. The aim of this study was to evaluate the effects of two CLAs (cis-9, trans-11 (CLA9,11) and trans-10, cis-12 (CLA10,12)) on the inflammatory responses of ECs. ECs (EA.hy926 cells) were cultured under standard conditions and exposed to CLAs (1 to 50 μM) for 48 h. Then, the cells were cultured for a further 6 or 24 h with tumour necrosis factor alpha (TNF-α, 1 ng/mL) as an inflammatory stimulant. ECs remained viable after treatments with 1 and 10 μM of each CLA, but not after treatment with 50 μM of CLA10,12. CLAs were incorporated into ECs in a concentration-dependent manner. CLA10,12 increased the levels of ICAM-1, IL-6, and RANTES in the culture medium, while CLA9,11 had null effects. Both CLAs (1 μM) decreased the appearance of NFκB1 mRNA, but only CLA9,11 maintained this downregulation at 10 μM. CLA10,12 had no effect on THP-1 cell adhesion to ECs while significantly decreasing the percentage of ECs expressing ICAM-1 and also levels of ICAM-1 expression per cell when used at 10 µM. Although CLA9,11 did not have any effect on ICAM-1 cell surface expression, it reduced THP-1 cell adhesion to the EA.hy926 cell monolayer at both concentrations. In summary, CLA10,12 showed some pro-inflammatory effects, while CLA9,11 exhibited null or anti-inflammatory effects. The results suggest that each CLA has different effects in ECs under a pro-inflammatory condition, highlighting the need to evaluate the effects of CLA isomers independently.     

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.     

Preparation, separation, and confirmation of the eight geometrical cis/trans conjugated linoleic acid isomers 8,10-through 11,13–18∶2

Conjugated linoleic acid (CLA) mixtures were isomerized with p-toluenesulfinic acid or I₂ catalyst. The resultant mixtures of the eight cis/trans geometric isomers of 8,10-, 9,11-, 10,12-, and 11,13-octadecadienoic (18∶2) acid methyl esters were separated by silver ion-high-performance liquid chromatography (Ag⁺-HPLC) and gas chromatography (GC). Ag⁺-HPLC allowed the separation of all positional CLA isomers and geometric cis/trans CLA isomers except 10,12–18∶2. However, one of the 8,10 isomers (8cis, 10trans-18∶2) coeluted with the 9trans,11cis18∶2 isomer. There were differences in the elution order of the pairs of geometric CLA isomers resolved by Ag⁺-HPLC. For the 8,10 and 9,11 CLA isomers, cis,trans eluted before trans,cis, whereas the opposite elution pattern was observed for the 11,13–18∶2 geometric isomers (trans,cis before cis,trans). All eight cis/trans CLA isomers were separated by GC on long polar capillary columns only when their relative concentrations were about equal. Large differences in the relative concentration of the CLA isomers found in natural products obscured the resolution and identification of a number of minor CLA isomers. In such cases, GC-mass spectrometry of the dimethyloxazoline derivatives was used to identify and confirm coeluting CLA isomers. For the same positional isomer, the cis,trans consistently eluted before the trans,cis CLA isomers by GC. High resolution mass spectrometry (MS) selected ion recording (SIR) of the molecular ions of the 18∶1 18∶2, and 18∶3 fatty acid methyl esters served as an independent and highly sensitive method to confirm CLA methyl ester peak assignments in GC chromatograms obtained from food samples by flame-ionization detection. The high-resolution MS data were used to correct for the nonselectivity of the flame-ionization detector.     

Thermally induced formation of conjugated isomers of linoleic acid

Chemical pathways responsible of the conjugation of linoleic acid during heat treatments such as refining (deodorization), frying or cooking processes have been investigated. For this purpose, methyl linoleate was submitted to oxidative and non‐oxidative thermal conditions. The resulting degradation products were mainly composed of geometrical and conjugated fatty acid isomers. Oxidative conditions were obtained using tert‐butyl hydroperoxide under inert atmosphere, and air. The obtained results from both thermal oxidative conditions were compared to non‐oxidative thermal treatment. Higher levels of conjugated linoleic acid were found when linoleate was heated under oxidative conditions. Two distinct mechanisms responsible for the formation of CLA isomers are proposed and discussed. Evidence of formation of 9,11‐C18:2 and 10,12‐C18:2 acids from 9,12‐C18:2 by a free‐radical chain reaction is provided. The first step consists in the formation of a free radical by abstraction of an active bis‐allylic hydrogen. By delocalization of the initial free radical, two allylic free radicals were stabilized and converted into the corresponding CLA isomers via the abstraction of a hydrogen radical from other linoleic acid or oxygenated species. Kinetic observations confirmed the significance of the bimolecular mechanism. Moreover, the proposed mechanism is supported by several pieces of information from the literature on peroxidation of linoleic acid. Under pure thermal conditions and/or for diluted samples, a second pathway to the formation of CLA from heat‐treated linoleic acid is proposed via an intramolecular rearrangement of the pentadienyl structure. This thermal [1,3]‐sigmatropic rearrangement results in a mixture of 9,11 and 10,12 CLA isomers. The formed cis/trans CLA isomers were readily rearranged by a [1,5]‐sigmatropic shift to yield trans‐8,cis‐10 and cis‐11,trans‐13 CLA isomers, respectively.     

Determination of CLA <Emphasis Type="Italic">trans</Emphasis>,<Emphasis Type="Italic">trans</Emphasis> Positional Isomerism in CLA-Rich Soy Oil by GC–MS and Silver Ion HPLC

A ~20% CLA-rich soy oil with low saturated fat and no cholesterol was produced by photoisomerizing soy oil linoleic acid. The oil is predominately trans,trans CLA, with the oil containing 17% trans,trans CLA. Recent studies have shown that trans,trans CLA-rich soy oil significantly reduces heart disease and diabetes risk factors in obese rats. However, the positional isomerism of these geometrical isomers has not been identified. The objectives of the study were to determine trans,trans CLA positional isomerism of CLA fatty acids in CLA-rich soy oil and determine the resolution of trans,trans CLA positional isomers by silver ion chromatography. GC–MS studies of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) derivatives of CLA-rich oil showed that 9,11 CLA and 10,12 CLA were the major positional isomers. These were hypothesized to be the trans,trans CLA isomers, which was confirmed by silver ion chromatography and subsequent GC–FID fatty acid and ATR-FTIR geometrical isomer analysis of the collected fractions. The identification of 9,11 trans,trans CLA and 10,12 trans,trans CLA as the major CLA isomers in CLA-rich oil then allowed the deduction of the synthetic mechanism of the photoisomerism of soy oil linoleic acid to trans,trans CLA.     

Occurrence of Conjugated Linolenic Acids in Purified Soybean Oil

A high-performance liquid chromatographic (HPLC) method is described for the determination of conjugated linoleic acids (CLA) and conjugated linolenic acids (CLN). Methyl esters prepared from purified lipid fractions of soybean oil were analyzed using an HPLC system equipped with photodiode-array detector to detect peaks having maximum absorption around 233 and 275 nm. These peaks were concentrated by AgNO₃-silicic acid column chromatography and reversed-phase HPLC. The structural analysis, of dimethyloxazoline (DMOX) derivatized methyl esters, using gas chromatography–mass spectrometry (GC–MS) showed the occurrence of 9,11- and 10,12-CLA and 8,10,13-, 8,10,12-, and 9,11,13-CLN. The comparison of these conjugated fatty acids with authentic isomers by HPLC revealed the presence of isomeric mixtures of CLA [cis (c),trans(t) or t,c and t,t] and CLN (c,t,t or t,t,c and t,t,t). Traces of 9,11- and 10,12-CLA (c,t or t,c) were found in crude oil. CLN isomers (8,10,12-18:3 and 9,11,13-18:3) were found to be forming during the bleaching phase of soybean oil processing. 8,10,13-CLN and 9,11- and 10,12-CLA (t,t) were only found in soybean oil after the deodorization step. CLN contents in commercial soybean oil varied from 387 to 1,316 mg/kg oil. A decreased level of bleaching earth and temperature resulted in a reduced CLN content. It is possible that CLN would be derived from the linoleate hydroperoxides formed during the processing and storage of soybean oil.     

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.     

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.     

Characterization of fatty acid isomers in dehydrated castor oil by gas chromatography and gas chromatography-mass spectrometry techniques

Fatty acid isomers present in dehydrated castor oil were analyzed by gas chromatography and gas chromatography-mass spectrometry (GC-MS) of their dimethyl oxazoline derivatives. Conjugated linoleic acid 9,11 and 10,12 isomers were identified by GC based on equivalent chainlengths. Segmental peak analysis of GC-MS total ion chromatogram mass fragmentation pattern revealed the presence of 7,9 and 8,10 conjugated linoleic acids along with 9,11 and 10,12 conjugated linoleic acids. Interestingly, a reverse elution of trans,trans-conjugated linoleic acids was observed, viz. 10,12 was followed by 9,11, which was in turn followed by the 8,10 and 7,9 isomers. The observed reverse order of elution was in contrast to the conventional elution pattern of both nonconjugated and conjugated cis,cis-, cis,trans- and trans,cis-isomers.     

Rosiglitazone,a PPAR-γ Agonist,Fails to Attenuate CLA-Induced Milk Fat Depression and Hepatic Lipid Accumulation in Lactating Mice

Our objective was to investigate the combination of rosiglitazone (ROSI) and conjugated linoleic acid (CLA) on mammary and hepatic lipogenesis in lactating C57Bl/6 J mice. Twenty-four lactating mice were randomly assigned to one of four treatments applied from postpartum day 6 to day 10. Treatments included: (1) control diet, (2) control plus 1.5 % dietary CLA (CLA) substituted for soybean oil, (3) control plus daily intra-peritoneal (IP) rosiglitazone injections (10 mg/kg body weight) (ROSI), and (4) CLA plus ROSI (CLA-ROSI). Dam food intake and milk fat concentration were depressed with CLA. However, no effects were observed with ROSI. The CLA-induced milk fat depression was due to reduced expression for mammary lipogenic genes involved in de-novo fatty acid (FA) synthesis, FA uptake and desaturation, and triacyglycerol synthesis. Liver weight (g/100 g body weight) was increased by CLA due to an increase in lipid accumulation triggering a compensatory reduction in mRNA abundance of hepatic lipogenic enzymes, including acetyl-CoA carboxylase I and stearoyl-CoA desaturase I. On the contrary, no effects were observed with ROSI on hepatic and mammary lipogenic gene and enzyme expression. Overall, feeding CLA to lactating mice induced milk fat depression and increased hepatic lipid accumulation, probably due to the presence of trans-10, cis-12 CLA isomer, while ROSI failed to significantly attenuate both hepatic steatosis and reduction in milk fat content.     

<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.     

Comparison of silver-ion high-performance liquid chromatographic quantification of free and methylated conjugated linoleic acids

Silver-ion high-performance liquid chromatography was used to fractionate a mixture of conjugated linoleic acid (CLA) isomers (as the free fatty acids, CLAFFA) in commercial CLA mixtures and biological samples. Due to the unchanged retention mechanism, it was assumed that the elution order of the isomers remained the same as that of methyl esters separated on the same column. The most abundant isomers, cis/trans 10,12-18:2 and cis/trans 9,11-18:2, were separated better as free acids on a single column than in the methyl ester form. Quantification of the CLA standard was used as the reference profile to evaluate different methylation methods commonly used to prepare CLA methyl esters for quantitation. Acid- and vuigi base-catalyzed derivatization methods resulted in CLA intraisomerization and losses in total conjugated dienes content. Acid (HCl and BF₃) methylations significantly elevated the level of trans,trans isomers and significantly reduced the cis/trans isomers. Base methylation, tetramethylguanidine/methanol, resulted in loss of trans,trans isomers, and a substantial loss of total underivatized conjugated dienes. Other catalysts such as the trimethylsilyldiazomethane produced additional peaks of unidentified artifacts. The analysis of CLAFFA appears to provide more accurate quantification of CLA isomers in commercial and biological samples.     

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

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

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.     

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.     

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.     

Supplementation with CLA: Isomer incorporation into serum lipids and effect on body fat of women

Animal studies have suggested that CLA, a natural component of meat and dairy products, may confer beneficial effects on health. However, human studies using supplementation with CLA have produced contradictory results. The aim of the present study was to further investigate the effect of CLA supplementation on human body fat, serum leptin, and serum lipids, as well as the incorporation of CLA isomers into serum lipids classes. Sixteen young healthy nonobese sedentary women received 2.1 g of CLA (divided equally between the cis,trans-9,11 and trans,cis-10,12 isomers) daily for 45 d and placebo for 45 d in a randomized double-blind crossover design. Body fat was estimated (by measurement of skinfold thickness at 10 sites), and blood was sampled at the beginning, middle, and end of the entire intervention period; an additional blood sample was obtained 2 wk thereafer. No significant differences in energy, carbohydrate, lipid, or protein intake existed between the CLA and placebo intake periods. No significant differences were found in body fat or serum leptin, TAG, total cholesterol, HDL-cholesterol, and alanine aminotransferase between CLA and placebo. The CLA isomer content of serum TAG, phospholipids, and total lipids increased 2–5 times with CLA supplementation (P<0.05). In contrast, the CLA content of cholesteryl esters did not change significantly. The period of 2 wk after the end of CLA supplementation was sufficient for its washout from serum lipids. These data indicate that supplementation with 2.1 g of CLA daily for 45 d increased its levels in blood but had no effect on body composition or the lipidemic profile of nonobese women.     

Influence of heat and refining on formation of CLA isomers in sunflower oil

The aims of this study were to determine whether CLA are formed during refining of vegetable oils and to study the level and composition of CLA during heating. The effects of three refining steps (neutralization, bleaching, and deodorization) were analyzed with respect to their effect on CLA content. Two temperatures (180 and 220°C) were used for heating; CLA appeared only after deodorization. The level of CLA was positively influenced by temperature. More CLA were present after treatment at 220°C than at 180°C (1.3 and 0.2% of total FA, respectively). The high temperature modified the relative proportions of the CLA isomers. The main CLA isomers in fresh or heated oils were the trans,trans ones (mainly 9,11 and 10,12 isomers).