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

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

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.     

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 cis-9,trans-11 CLA in rats at intake levels reported for breast-fed infants

CLA intake in exclusively breast-fed infants is close to levels found to have physiological effects in animals. However, in the majority of studies mixtures of CLA isomers have been used and the independent effects of the major CLA isomer in human milk, cis-9, trans-11 CLA, at the intake level in exclusively breast-fed infants have hardly been studied. We therefore studied the effects of cis-9, trans-11 CLA on plasma lipids and glucose, immune function, and bone metabolism in growing rats. Thirty male Sprague-Dawley rats (n=10/group) were fed either 20 mg/kg/d cis-9, trans-11 CLA and 20 mg/kg/d sunflower oil (CLA20), 40 mg/kg/d cis-9, trans-11 CLA (CLA40), or 40 mg/kg/d sunflower oil (placebo) for 8 wk. No significant differences between groups were found in plasma lipids, glucose, insulin, C-reactive protein, or lipid peroxidation. Liver fat content was lowest in the CLA20 group. In vitro interleukin 2 (IL-2) production increased, and tumor necrosis factor alpha, IL-1β, prostaglandin E₂, and leukotriene B₄ production decreased in the CLA20 group. No differences between groups were detected in IL-4, IL-6, or interferon gamma production, plasma osteocalcin, insulin-like growth factor, or urinary deoxypyridino line crosslinks. Plasma tartrate-resistant acid phosphatase 5b activity was significantly increased in the CLA40 group. The results indicate anti-inflammatory effects and enhanced T-cell function for the CLA20 group. No adverse effects were seen in the CLA20 group, whereas indications of increased bone resorption rate were observed in the CLA40 group.     

A simple method of preparation of methyl trans-10,cis-12- and cis-9,trans-11-octadecadienoates from methyl linoleate

Pure conjugated isomers of linoleic acid were prepared on a large scale by alkali-isomerization of purified methyl linoleate. The methyl esters of alkali-isomerized linoleic acid contained mainly the methyl cis-9,trans-11- and trans-10,cis-12-octadecadienoates (44 and 47%, respectively). These two isomers were then separated and purified by a series of low-temperature crystallizations from acetone. The isomeric purity obtained for the cis-9,trans-11-octadecadienoate isomer was >90% and that of the trans-10,cis-12-octadecadienoate isomer was 89 to 97%. The isolated yield of the two isomers corresponded to 18 and 25.7%, respectively, of the starting material. The structure of the two isomers was confirmed using partial hydrazine reduction, silver nitrate-thin-layer chromatography of the resulting monoenes and gas chromatography coupled with mass spectrometry of the 4,4-dimethyloxazoline derivatives. Fourier transform infrared spectroscopy of the monoenes gave the confirmation of the geometry of each double bond.     

Infrared absorption of methylcis-9,trans-11-, andtrans-10,cis-12-octadecadienoates

The ratio of absorptivity at 10.2 µm and 10.6 µm differs between methylcis-9,trans-11-, andtrans-10,cis-12-octadecadienoates. For thecis-9,trans-11-ester, a_10.2 µm/a_10.6 µm is in the range of 1.1–1.2; for thetrans-10,cis-12-ester, it is 1.3–1.4. These differences in absorptivities are great enough to affect significantly compositions calculated from IR absorption.     

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.     

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

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

A new acid fromcalea urticaefolia seed oil:trans-3,cis-9,cis-12-octadecatrienoic acid

A major constituent fatty acid (31.2%) fromCalea urticaefolia (Mill.) DC. seed oil is the previously unknowntrans-3,cis-9,cis-12-octadecatrienoic acid. The oil also contains 2.2% of an unidentified acid and others with gas-liquid chromatographic characteristics that correspond to the conventional fatty acids: myristic, 0.1%; palmitic, 9.3%; stearic, 2.9%; oleic, 5.3%; and linoleic, 48.9%. Presented at the AOCS Meeting in New Orleans, 1964. No. Utiliz. Res. & Dev. Div., ARS, USDA.     

Desaturation indices in liver, muscle, and bone of growing male and female mice fed trans-10, cis-12 conjugated linoleic acid

trans-10, cis-12-CLA (t10,c12-CLA) inhibits lipid deposition in adipose tissue of many species, but it also enhances lipid deposition in liver. We evaluated effects of dietary t10,c12-CLA content and gender on carcass composition, FA profile of selected tissues, and expression of FA synthase (FAS) and stearoyl-CoA desaturase-1 (SCD) mRNA in adipose tissue. Male and female (63 of each) CD-1 mice were assigned a diet containing 0.0, 0.15, or 0.30% t10,c12-CLA at 4 wk of age. Seven mice per dietary group within gender were sacrificed after 2,4 or 6 wk. The CLA isomer caused dose-dependent reductions in dry carcass weight and fat content, without altering protein content, but carcass fat and epididymal fat pad weights of males were reduced to a greater extent than carcass fat and inguinal fat pad weights of females. FAS and SCD mRNA in adipose tissue was more abundant in females than males, but expression in both genders decreased as the t10, c12-CLA content of the diet increased. Although the weight of gastrocnemius muscle was not influenced by diet, total FA content of the muscle of both genders decreased in response to dietary t10,c12-CLA content. Femur weight of male mice increased as the t10,c12-CLA content of the diet increased, but the weight increase was associated with a reduction in total FA content. The Δ9 desaturation indices for muscle and femur suggested a linear reduction in SCD activity, whereas Δ9 indices for liver indicated linear enhancement of SCD activity. Overall, results suggested that growing male mice were more susceptible than females to t10,c12-CLA inhibition of lipid deposition.     

Isomerization of 9c11t/10t12c CLA in Triacylglycerols

Isomers of conjugated linoleic acid from 7t9c through 12t14t can be induced by thermal treatment of triacylglycerol samples of 9c11t or 10t12c fatty acids in glass tubes. The formation of conjugated linoleic acids (CLAs) has been observed during thermal induction of the above-mentioned triacylglycerols at 250, 280 and 320°C. The concentrations of isomers formed in the mixture varied depending on the temperature and duration of the heating experiments. The objective of this study was to find a suitable thermal induction temperature and time that can produce most of the isomers of CLAs from the above-mentioned triacylglycerols. Such a mixture would give researchers a reference standard that can be used in the identification of CLAs in GC analyses of relevant samples. Fifteen-microlitre portions of the triacylglycerol samples containing 9c11t/10t12c fatty acid were placed in micro-glass ampoules, sealed under nitrogen and then subjected to thermal treatment. The glass ampoules were removed at regular time intervals, cut open, and part of the samples was analysed by infrared spectroscopy using attenuated internal reflectance technique. The remainder of the samples was subjected to derivatisation into their methyl esters. The methyl esters of the isomerised fatty acids were then analysed by gas chromatography after appropriate dilution in heptane. The results show that the thermally induced glyceride samples of 9c11t/10t12c fatty acids gave CLA profiles containing isomers ranging from 7t9c to 12t14t. However, the concentrations of the isomers are different depending on the duration of the thermal induction. It appears that [1,5] sigmatropic rearrangements and positional isomerisations take place in the heated mixtures. The rearrangements and positional isomerisations are accelerated by increasing temperature. The glyceride samples heated to 325°C form isomers within 30 min and provide a mixture of CLA isomers that can be used as reference sample containing the methyl esters of CLAs.     

High Dose trans-10,cis-12 CLA Increases Lean Body Mass in Hamsters, but Elevates Levels of Plasma Lipids and Liver Enzyme Biomarkers

The current study examined the efficacy of graded doses of c9,t11 and t10,c12 CLA isomers on body composition, energy expenditure, hepatic and serum lipid liver biomarkers in hamsters. Animals (n = 105) were randomized to seven treatments (control, 1, 2, 3% of c9,t11; 1, 2, 3% of t10,c12) for 28 days. After 28 days treatment, 1–3% of t10,c12 lowered (p < 0.05) body fat mass compared to the control group. The 1–3% t10,c12 and 3% c9,t11 fed groups showed higher (p < 0.05) lean mass compared to other groups. We observed unfavorable changes in plasma total cholesterol and non-HDL cholesterol levels in animals fed with 3% t10,c12 CLA isomers. The 2%, 3% t10,c12 groups presented elevated (p < 0.05) ALT levels. The present data suggest that a diet enriched with more than 2% t10, c12 led to liver malfunction and poses unfavorable changes on plasma lipid profiles. The 1% t10,c12 CLA lowered (p < 0.05) body fat mass and increased (p < 0.05) lean body mass. The c9,t11 CLA has less potent actions than t10,c12 CLA. We conclude that the actions of CLA on energy and lipid metabolism are form and dose dependent in the hamster model.     

Large-scale synthesis of methyl cis-9, trans-11-octadecadienoate from methyl ricinoleate

The conjugated linoleic acid methyl cis-9,trans-11-octadecadienoate has been prepared on a large scale from methyl ricinoleate. Methyl ricinoleate was purified from castor esters by a partition method. It was converted to the mesylate, which was reacted with a base (1,8-diazabicyclo[5,4,0]-undec-7-ene) to give a product that contained 66% of the desired ester. Two urea crystallizations produced a product containing 83% methyl cis-9,trans-11-octadecadienoate, the identity of which was confirmed by gas chromatography linked to mass spectrometry and by Fourier transform infrared spectroscopy. The remaining impurities were methyl cis-9,cis-11- and cis-9-,trans-12-octadecadienoate.     

Lipoxygenase fromZea mays: 9-d-hydroperoxy-trans-10,cis-12-octadecadienoic acid from linoleic acid

Lipoxygenase from the germ of corn,Zea mays, oxidized linoleic acid to primarily 9-d-hydroperoxy-trans-10,cis-12-octadecadienoic acid. No. Utiliz. Res. Dev. Div., ARS, USDA.     

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.     

Evidence that commercial calf and horse sera can contain substantial amounts of trans-10,cis-12 conjugated linoleic acid

We analyzed fetal calf, newborn calf, horse, and adult cow sera for conjugated linoleic acid (CLA). All sera samples contained CLA, but the amounts varied. The predominant isomer was cis-9,trans-11 CLA but some samples appeared to contain substantial amounts of an isomer with the retention time of trans-10,cis-12 CLA.     

Dietary sandalwood seed oil modifies fatty acid composition of mouse adipose tissue, brain, and liver

Sandalwood (Santalum spicatum) seed oil, which occurs to about 50% of the weight of the seed kernels, contains 30–35% of total fatty acids (FA) as ximenynic acid (XMYA). This study was designed to obtain basic information on changes in tissue FA composition and on the metabolic fate of XMYA in mice fed a sandalwood seed oil (SWSO)-enriched diet. Female mice were randomly divided into three groups, each receiving different semisynthetic diets containing 5.2% (w/w) fat (standard laboratory diet), 15% canola oil, or 15% SWSO for 8 wk. The effects of SWSO as a dietary fat on the FA composition of adipose tissue, brain, and liver lipids were determined by analyses of FA methyl ester derivatives of extracted total lipid. The FA compositions of the liver and adipose tissue were markedly altered by the dietary fats, and mice fed on a SWSO-enriched diet were found to contain XMYA but only in low concentration (0.3 3%) in these tissues; XMYA was not detected in brain. Oleic acid was suggested to be a principal XMYA biotransformation product. The results were interpreted to suggest that the metabolism of XMYA may involve both biohydrogenation and oxidation reactions.     

A soy extract catalyzes formation of 9-oxo-trans-12,13-epoxy-trans-10-octadecenoic acid from 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid

In the presence of oxygen, a crude soy extract converted 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid into numerous products, from which 9-oxo-trans-12,13-epoxy-trans-10-octadecenoic acid was isolated. Additionally, the soy extract oxidized linoleic acid to the oxo-epoxyoctadecenoic acid, presumably via a sequential reaction involving lipoxygenase oxidation of linoleic acid followed by degradation of the resultant linoleic acid hydroperoxide. However, the linoleic acid substrate yielded two isomeric linoleic acid hydroperoxides and because of this, two isomeric oxoepoxyoctadecenoic acids. Presented in part at the 13th Congress, International Society for Fat Research, Marseilles, France, August 30–September 4, 1976.     

Cis-trans isomerization of octadecatrienoic acids during heating. Study of pinolenic (cis-5,cis-9,cis-12 18∶3) acid geometrical isomers in heated pine seed oil

To understand the heat-inducedcis-trans isomerization of ethylenic bonds in octadecatrienoic acids, pine seed oil, which contains the unusual nonmethylene-interrupted pinolenic (cis-5,cis-9,cis-12 18∶3) acid as a major component, was heated under vacuum at 240°C for 6 h together with linseed and borage oils. As a results, a small percentage of pinolenic acid undergoescis-trans isomerization. The main isomer that accumulates is thetrans-5,cis-9,trans-12 18∶3 acid. Minor amounts of the three mono-trans isomers are also present. Identification of isomers was realized by combining gas-liquid chromatography on a CP Sil 88 capillary column, argentation thin-layer chromatography and comparing the equivalent chainlengths of artifacts to those of isomers present in NO₂-isomerized pine seed oil. Hydrazine reduction was used to demonstrate that there was no positional shift of double bonds. Heat-induced geometrical isomerization of pinolenic acid differs from that of α- and γ-linolenic acids in at least two aspects. The reaction rate is slower (about one-fourth), and mono-trans isomers are formed in low amounts.