The biologically active isomers of conjugated linoleic acid.

Numerous physiological effects are attributed to conjugated linoleic acid (CLA). The purpose of this presentation is to consider these effects with respect to the cis-9,trans-11 and trans-10,cis-12 CLA isomers. We review previously published data and present new findings that relate to underlying biochemical mechanisms of action. Both isomers are natural products. The cis-9,trans-11 isomer is the principal dietary form of CLA, but the concentrations of this isomer and the trans-10,cis-12 isomer in dairy products or beef vary depending on the diet fed to cows or steers, respectively. The trans-10,cis-12 CLA isomer exerts specific effects on adipocytes, in particular reducing the uptake of lipid by inhibiting the activities of lipoprotein lipase and stearoyl-CoA desaturase. The trans-10,cis-12 CLA isomer also affects lipid metabolism in cultured Hep-G2 human liver cells, whereas both the cis-9,trans-11 and trans-10,cis-12 CLA isomers appear to be active in inhibiting carcinogenesis in animal models. We present new findings indicating that the cis-9,trans-11 CLA isomer enhances growth and probably feed efficiency in young rodents. Accordingly, the effects of CLA on body composition (induced by trans-10,cis-12 CLA) and growth/feed efficiency (induced by cis-9,trans-11 CLA) appear to be due to separate biochemical mechanisms. We also show that a 19-carbon CLA cognate (conjugated nonadecadienoic acid, CNA) inhibits lipoprotein lipase activity as effectively as CLA in cultured 3T3-L1 adipocytes. Presumably, CNA is metabolized differently than the 18-carbon CLA isomers, so this finding indicates direct activity of the administered compound as opposed to acting via a metabolite.     

Structural analysis of conjugated linoleic acid produced by Lactobacillus plantarum,and factors affecting isomer production

An isomer of the conjugated linoleic acid (CLA) produced from linoleic acid by Lactobacillus plantarum was identified as cis-9,trans-11-octadecadienoic acid by proton nuclear magnetic resonance spectroscopy. Together with earlier results, we concluded that the bacterium produces two CLA isomers, cis-9,trans-11- and trans-9,trans-11-octadecadienoic acid from linoleic acid. The addition of L-serine, glucose, AgNO3, or NaCl to the reaction mixture reduced production of the latter.     

Inhibition of hepatic stearoyl-CoA desaturase activity by trans-10, cis-12 conjugated linoleic acid and its derivatives

Conjugated linoleic acid (CLA) has been reported to decrease stearoyl-CoA desaturase (SCD) activity by decreasing mRNA expression. This investigation was designed to determine whether structurally related compounds of CLA have a direct inhibitory effect on SCD activity. Trans-10,cis-12 CLA had strong inhibitory activity on SCD while cis-9,trans-11, and trans-9,trans-11 isomers had no effect. Trans-10 octadecenoate was not inhibitory, whereas cis-12 octadecenate was inhibitory, but not as effective as trans-10,cis-12 CLA. Of the oxygenated derivatives, 9-peroxy-cis/trans-10, trans-12 octadecadienoate was a more effective inhibitor than trans-10,cis-12 CLA, whereas 9-hydroxy-trans-10, cis-12 octadecadienoate was less effective. Interestingly, cis-11 octadecadienoate and cis-12 octadecen-10-ynoate were slightly inhibitory. However, trans-9 and trans-11 octadecenoates, and trans-9,cis-12 octadecadienoate were all inactive under test condition, as were linoleate, oleate, and arachidonate. Derivatives of CLA acid modified to alcohol, amide or chloride were all inactive. A cis-12 double bond appears to be a key structural feature for inhibiting SCD activity, especially when coupled with a trans-10 double, whereas a cis-11 double bond is less effective.     

Biohydrogenation of unsaturated fatty acids. Presence of dithionite and an endogenous electron donor in Butyrivibrio fibrisolvens.

Two oxygen-consuming substances were isolated from cell-free extracts of the rumen anaerobe, Butyrivibrio fibrisolvens. The major fraction comprising 97% of the total activity was characterized as a three-component mixture of glucose, maltose, and dithionite. The minor activity fraction contained an electron donor for the reduction of cis-9,trans-11-octadecadienoate to trans-11-octadecenoate. After oxidation, the electron donor could be reduced by the dithionite, thereby accounting for the previously observed capacity of cell-free extracts of the bacterium to carry out the biohydrogenation of the conjugated dienoic fatty acid.     

Synthesis of [17,18-3H] trans-13-azaprostanoic acid. A labeled probe for the PGH2/TXA2 receptor

Because of its highly unstable nature, TXA2, produced by platelet metabolism of arachidonic acid, does not lend itself to use as a receptor probe for its own receptor. As such, the stable TXA2/PGH2 antagonist, trans-13-azaprostanoic acid (trans-13-APA, 12b), was prepared as the [17, 18 3H] derivative [( 3H] trans-13-APA, 12c) to study this receptor and to better evaluate the mechanism of action of these azaprostanoids. Tritiated trans-13-APA, 12c, was prepared in nearly theoretical specific activity (57 Ci/mmole) from (17Z)-trans-13-azaprost-17-enoic acid (11b) by catalytic tritiation. The unsaturated 11b was prepared by condensation of cis-7-amino-3-heptene (8) with 2-(6-carboxyhexyl) cyclopentanone (9), NaBH4 reduction, chromatography, and hydrolysis of the trans isomer so isolated. The olefins 11a and b were also of biochemical interest because of the unsaturation in the lower side chain. The presence of similar unsaturation in PGH3(4) and TXA3 (3) renders these prostaglandins inactive as proaggregatory agents. Evaluation of the antiaggregatory activity of 11a and b indicated it to be about the same potency in inhibiting human platelet aggregation as the parent cis and trans-13-APAs, suggesting that introduction of a double bond at the 17 position in platelet prostaglandin antagonists is unlikely to result in enhanced antiplatelet activity.     

Conjugated linoleic acids promote human fat cell apoptosis.

Conjugated linoleic acids (CLAs) are conjugated dienoic isomers of linoleic acid. Some isomers have been shown to reduce fat mass in animal and cell culture models. However, controversial results were obtained in studies of supplementation of CLAs in human subjects. In order to get more insights into the direct effects of CLAs on human fat cells, we have studied the influence of cis-9, trans-11 CLA and trans-10, cis-12 CLA on the biology of human SGBS preadipocytes and adipocytes. Both CLA isomers equally inhibited the proliferation of preadipocytes in a dose-dependent manner. Continuous treatment with 1-10 microM trans-10, cis-12 CLA, and to a weaker extent cis-9, trans-11 CLA, inhibited accumulation of lipids during adipogenic differentiation. Treatment with higher doses of CLA induced apoptosis in preadipocytes, in differentiating cells, and adipocytes. The trans-10, cis-12 isomer had a higher apoptotic potency in adipocytes than cis-9, trans-11 CLA. Taken together, the treatment of human preadipocytes and adipocytes with physiological relevant concentrations of CLAs resulted in an impairment of proliferation and differentiation and induction of apoptosis. The trans-10, cis-12 isomer was more potent than the cis-9, trans-11 isomer. Further clinical studies are needed to evaluate the effects of CLAs on human fat mass and metabolism in vivo.     

New conjugated hydroxydienoic fatty acids and acetotriacylglycerols from Securidaca longipedunculata seed oil.

Like other members of the plant family Polygalaceae, Securidaca longipedunculata Fres., is a source of fatty acids and triacylglycerols with unusual structures. Its seed oil contains at least seven chromatographically distinct groups of triacylglycerols divided into two series: One series represents monoacetotriacylglycerols, and the other 'normal' triacylglycerols having only long-chain fatty acids. Each series has groups containing zero, one or two conjugated hydroxydienoic acids. In addition, there is a small amount of triacylglycerol incorporating three hydroxy acids. In addition to coriolic (13-hydroxyoctadeca-cis-9,trans-11-dienoic) acid (27%), two of its previously unknown homologs are present: 11-hydroxyhexadeca-cis-7,trans-9-dienoic acid (15%) and 9-hydroxytetradeca-cis-5,trans-7-dienoic acid (2%).     

Co-oxidation of carotenes requires one soybean lipoxygenase isoenzyme.

The type II lipoxygenase (optimum pH 6.5) from soybeans was purified and separated into two fractions either by chromatography on DEAE-Sephadex or by isoelectric focusing. In the presence of linoleic acid and oxygen both fractions co-oxidise canthaxanthine or beta-carotene as effectively as a combination of these fractions. Oxygenation of linoleic acid and co-oxidation of canthaxanthine by type II lipoxygenase is stimulated by 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid but not by 13-hydroxy-cis-9,trans-11-octadecadienoic acid or 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid.     

Ricinoleic acid and castor oil as substrates for conjugated linoleic acid production by washed cells of Lactobacillus plantarum

Ricinoleic acid (12-hydroxy-cis-9-octadecaenoic acid) was an effective substrate for conjugated linoleic acid (CLA) production by washed cells of Lactobacillus plantarum AKU 1009a. The CLA produced was a mixture of cis-9,trans-11- and trans-9,trans-11-octadecadienoic acids. Addition of alpha-linolenic acid to the culture medium increased the CLA productivity of the washed cells. In the presence of lipase, castor oil, in which the main fatty acid component is ricinoleic acid, also was a substrate for CLA.     

The role of Delta(9)-desaturase in the production of cis-9, trans-11 CLA

Biomedical studies with animal models have demonstrated that cis-9, trans-11 conjugated linoleic acid (CLA), the predominant isomer found in milk fat from dairy cows, has anticarcinogenic effects. We recently demonstrated endogenous synthesis of cis-9, trans-11 CLA from ruminally derived trans-11 C18:1 by Delta(9)-desaturase in lactating dairy cows. The present study further examined endogenous synthesis of cis-9, trans-11 CLA and quantified its importance by increasing substrate supply using partially hydrogenated vegetable oil (PHVO) as a source of trans-11 C18:1 and blocking endogenous synthesis using sterculic oil (SO) as a source of cyclopropene fatty acids which specifically inhibit Delta(9)-desaturase. Four cows were abomasally infused with 1) control, 2) PHVO, 3) SO, and 4) PHVO+SO in a 4 x 4 Latin square design. With infusion of PHVO, cis-9, trans-11 CLA was increased by 17% in milk fat. Consistent with inhibition of desaturase, SO treatments increased milk fat ratios for the fatty acid pairs effected by Delta(9)-desaturase, C14:0/cis-9 C14:1, C16:0/cis-9 C16:1, and C18:0/cis-9 C18:1. The role of endogenous synthesis of CLA was evident from the 60-65% reduction in cis-9, trans-11 CLA which occurred in milk fat with SO treatments. cis-9 C14:1 originates from desaturation of C14:0 by Delta(9)-desaturase and can be used to estimate the extent of SO inhibition of Delta(9)-desaturase. When this correction factor was applied, endogenous synthesis was estimated to account for 78% of the total cis-9, trans-11 CLA in milk fat. Thus, endogenous synthesis was the major source of cis-9, trans-11 CLA in milk fat of lactating cows.     

Isomers of conjugated linoleic acids are synthesized via different mechanisms in ruminal digesta and bacteria

Digesta samples from the ovine rumen and pure ruminal bacteria were incubated with linoleic acid (LA) in deuterium oxide-containing buffer to investigate the mechanisms of the formation of conjugated linoleic acids (CLAs). Rumenic acid (RA; cis-9,trans-11-18:2), trans-9,trans-11-18:2, and trans-10,cis-12-18:2 were the major CLA intermediates formed from LA in ruminal digesta, with traces of trans-9,cis-11-18:2, cis-9,cis-11-18:2, and cis-10,cis-12-18:2. Mass spectrometry indicated an increase in the n+1 isotopomers of RA and other 9,11-CLA isomers, as a result of labeling at C-13, whereas 10,12 isomers contained minimal enrichment. In pure culture, Butyrivibrio fibrisolvens and Clostridium proteoclasticum produced mostly RA with minor amounts of other 9,11 isomers, all labeled at C-13. Increasing the deuterium enrichment in water led to an isotope effect, whereby (1)H was incorporated in preference to (2)H. In contrast, the type strain and a ruminal isolate of Propionibacterium acnes produced trans-10,cis-12-18:2 and other 10,12 isomers that were minimally labeled. Incubations with ruminal digesta provided no support for ricinoleic acid (12-OH,cis-9-18:1) as an intermediate of RA synthesis. We conclude that geometric isomers of 10,12-CLA are synthesized by a mechanism that differs from the synthesis of 9,11 isomers, the latter possibly initiated by hydrogen abstraction on C-11 catalyzed by a radical intermediate enzyme.     

Conjugated linoleic acid accumulation via 10-hydroxy-12-octadecaenoic acid during microaerobic transformation of linoleic acid by Lactobacillus acidophilus

Specific isomers of conjugated linoleic acid (CLA), a fatty acid with potentially beneficial physiological and anticarcinogenic effects, were efficiently produced from linoleic acid by washed cells of Lactobacillus acidophilus AKU 1137 under microaerobic conditions, and the metabolic pathway of CLA production from linoleic acid is explained for the first time. The CLA isomers produced were identified as cis-9, trans-11- or trans-9, cis-11-octadecadienoic acid and trans-9, trans-11-octadecadienoic acid. Preceding the production of CLA, hydroxy fatty acids identified as 10-hydroxy-cis-12-octadecaenoic acid and 10-hydroxy-trans-12-octadecaenoic acid had accumulated. The isolated 10-hydroxy-cis-12-octadecaenoic acid was transformed into CLA during incubation with washed cells of L. acidophilus, suggesting that this hydroxy fatty acid is one of the intermediates of CLA production from linoleic acid. The washed cells of L. acidophilus producing high levels of CLA were obtained by cultivation in a medium containing linoleic acid, indicating that the enzyme system for CLA production is induced by linoleic acid. After 4 days of reaction with these washed cells, more than 95% of the added linoleic acid (5 mg/ml) was transformed into CLA, and the CLA content in total fatty acids recovered exceeded 80% (wt/wt). Almost all of the CLA produced was in the cells or was associated with the cells as free fatty acid.     

The PMR analysis of non-conjugated alkenoic and alkynoic acids and esters.

The 220 MHz PMR spectra of 143 non-conjugated alkenoic and alkynoic acids and esters are correlated so as to provide a method for the structural analysis of such compounds in general. The spectral data are explained in terms of long-range deshielding of the double bonds, triple bonds, acid and ester groups in the molecules, and parameters are derived to quantify the influence of these groups on the chemical shifts of methyl and methylene protons up to six carbon atoms distant along an alkyl chain. It is shown that, by the application of these parameters, 220 MHz PMR spectroscopy can be used to determine both the stereochemistry and position of double bonds, and the position of triple bonds, in the majority of fatty acids and esters. The 2- to 9- and 13- to 17-cis- and trans-isomers of octadecenoic acid may be readily idenfited in this way, whilst for the octadecynoic acids all positional isomers may be characterized. Examples are also given of the structural analysis of several polyenoic compounds, including methyl cis-5, cis-8, cis-11, cis-14, cis-5, cis-8, cis-11, trans-14, and trans-5, cis-8, cis-11, cis-14-eicosatetraenoates, and methyl trans-5, cis-9, cis-12-octadecatrienoate.     

Effect of conjugated linoleic acid isomers on lipoproteins and atherosclerosis in the Syrian Golden hamster

Conjugated linoleic acid (CLA) is a group of positional and geometric isomers of linoleic acid (LA, C18:2 cis-9, cis-12) that are reported to have important biological activities, including protection against atherosclerosis. In this study, the potential role of the individual cis-9, trans-11 and trans-10, cis-12 isomers of CLA in atherogenesis were compared with LA in the Syrian Golden hamster. Supplementation of a high-fat, high-cholesterol diet (HFHC) with 1% (w/w) cis-9, trans-11 CLA or trans-10, cis-12 CLA did not significantly affect plasma cholesterol levels compared to supplementation with 1% (w/w) LA. Very low density lipoprotein cholesterol (VLDL-C) was lower and plasma triglycerides (TG) were higher in diets where C18:2 fatty acid was added to the HFHC diet, but neither the cis-9, trans-11 CLA group nor trans-10, cis-12 CLA group was significantly different from the LA control group. CLA supplementation did not significantly affect low density lipoprotein cholesterol (LDL-C). Trans-10, cis-12 CLA increased high density lipoprotein cholesterol (HDL-C) levels compared to LA or cis-9, trans-11 CLA (P<0.02), and although the ratio of non-HDL-C:HDL-C in the cis-9, trans-11 CLA group (1.11+/-0.54) and the trans-10, cis-12 CLA group (1.11+/-0.21) was lower than the LA group (1.29+/-0.45), the reduction did not reach statistical significance. Atherosclerosis was assessed in the ascending aorta by measuring the number of aortic cross-sections containing Oil Red O-stained intimal lesions. Compared to the LA group (60+/-11%), both the cis-9, trans-11 CLA group (38+/-8%) and the trans-10, cis-12 CLA group (28+/-7%) had fewer sections displaying a fatty streak lesion, although the differences did not reach statistical significance. These results suggest that individual CLA isomers may reduce atherosclerotic lesion development in the hamster, but when compared to LA, the apparent atheroprotective effects do not correlate with beneficial changes in lipoprotein profile.     

Cis and trans conformational changes of bacterial fatty acids in comparison with analogs of animal and vegetable origin

The conditions of the formations of trans isomers of fatty acids, depending on the method of processing and storage of the raw material of microbial, plant and animal origin, were investigated. In the composition of lipids, except for the main trans-isomer elaidic acid, nonsignificant amounts of trans -2-hexen-4-ynal, trans-2-formlcyclopro-panecarboxylate, methyl octadeca-9-yn-l1-trans-enoate, trans-2, 2-dimethyl-3-(2-propenyl)-ethyl ester, trans-9-octadecenoic acid, and trans-1,5-heptadiene, and mixed isomers of methyloctadeca-9-yn-11-trans-enoate,-methyl-9-cis, 11-trans-octadecadienoate, l-[trans-4-(2-iodo-ethyl) cyclohexyl]-trans-4-pentylcyclo-hexane and cis-9, and trans 11-octadecenoic acid. The major trans elaidic acid component was detected in natural objects of different origin in quantities not exceeding 0.05–0.11%. The combination of thermal processing with other parameters, especially enzymatic treatment, led to an increased proportion of trans isomers. The content of trans isomers is usually proportional to the time of storage of materials.     

Trans-10, cis-12, but not cis-9, trans-11 CLA isomer,inhibits brown adipocyte thermogenic capacity

Conjugated linoleic acid (CLA) is reported to have health benefits, including reduction of body fat. Previous studies have shown that brown adipose tissue (BAT) is particularly sensitive to CLA-supplemented diet feeding. Most of them use mixtures containing several CLA isomers, mainly cis-9, trans-11 and trans-10, cis-12 in equal concentration. Our aim was to characterize the separate effects of both CLA isomers on thermogenic capacity in cultured brown adipocytes. The CLA isomers showed opposite effects. Hence, on the one hand, trans-10, cis-12 inhibited uncoupling protein (UCP) 1 induction by norepinephrine (NE) and produced a decrease in leptin mRNA levels. These effects were associated with a blockage of CCAAT-enhancer-binding protein-alpha and peroxisome proliferator-activated receptor-gamma(2) mRNA expression. On the other hand, cis-9, trans-11 enhanced the UCP1 elicited by NE, an effect reported earlier for polyunsaturated fatty acids and also observed here for linoleic acid. These findings could explain, at least in part, the effects observed in vivo when feeding a CLA mixture supplemented diet as a result of the combined action of CLA isomers (reduction of adipogenesis and defective BAT thermogenesis that could be through trans-10, cis-12 and enhanced UCP1 thermogenic capacity through cis-9, trans-11).     

Inhibition of stearoyl-CoA desaturase activity by the cis-9,trans-11 isomer and the trans-10,cis-12 isomer of conjugated linoleic acid in MDA-MB-231 and MCF-7 human breast cancer cells

Conjugated linoleic acid (CLA) is a collective term for a group of positional and geometric conjugated dienoic isomers of linoleic acid. CLA has been shown to have strong inhibitory effects on mammary carcinogenesis both in vitro and in vivo. In this study, we investigated the regulation of human stearoyl-CoA desaturase (SCD, EC 1.14.99.5) expression by CLA in human breast cancer cell lines, MDA-MB-231 and MCF-7. Treatment of the cells with the cis-9,trans-11 and trans-10,cis-12 CLA isomers (45 microM) did not repress SCD mRNA in both MDA-MB-231 and MCF-7 cells. However, the cis-9,trans-11 and trans-10,cis-12 CLA isomers significantly decreased SCD protein levels and SCD activity in MDA-MB-231 cells. In MCF-7 cells, both isomers did not affect protein levels, but they inhibited SCD activity. These results suggest that in MDA-MB-231 cells the cis-9,trans-11 and trans-10,cis-12 CLA isomers regulate human SCD by reducing SCD protein levels, while in MCF-7 cells both isomers have a direct inhibitory effect on SCD enzyme activity.     

Reduction of linoleic acid inhibition in production of conjugated linoleic acid by Propionibacterium freudenreichii ssp. shermanii

A method for the production of conjugated linoleic acid (CLA) from linoleic acid (LA) using growing cultures of Propionibacterium freudenreichii ssp. shermanii JS was developed. The growth inhibitory effect of LA was eliminated by dispersing it in a sufficient concentration of polyoxyethylene sorbitan monooleate detergent. For the whey permeate medium used, the optimum LA:detergent ratio was 1:15 (w/w). As a result, the cultures tolerated at least 1000 microg x mL(-1) LA, which was converted to CLA with 57%-87% efficiency. The cis-9, trans-11 and trans-9, cis-11 isomers constituted 85%-90% of the CLA produced. The feasibility of the method was demonstrated also in de Man Rogosa-Sharpe (MRS) broth.     

Metabolism of linoleic acid by human gut bacteria: different routes for biosynthesis of conjugated linoleic acid

A survey of 30 representative strains of human gram-positive intestinal bacteria indicated that Roseburia species were among the most active in metabolizing linoleic acid (cis-9,cis-12-18:2). Different Roseburia spp. formed either vaccenic acid (trans-11-18:1) or a 10-hydroxy-18:1; these compounds are precursors of the health-promoting conjugated linoleic acid cis-9,trans-11-18:2 in human tissues and the intestine, respectively.     

Determination of double bond positions in fatty acids with conjugated double bonds

The positions of the double bonds in fatty acids with conjugated double bonds may be determined by mass spectrometry of the methyl esters of their trimethylsilyl ether derivatives obtained by hydroxylation of the double bonds followed by silylation of the resulting polyols. The method has been applied to trans-9,trans-11- and trans-10, trans-12-octadecadienoic acid.