We evaluated the effect of cis-9, trans-11 (9c,11t) and trans-10, cis-12 (10t,12c) conjugated linoleic acid (CLA) on the immune system in C57BL/6J mice. Mice were fed experimental diets containing 0% CLA (controls), 1% 9c,11t-CLA, 1% 10t,12c-CLA or a 1:1 mixture (0.5% + 0.5%) of these two CLA isomers for 3 wk. Relative spleen weights of all CLA fed mice were greater than the controls. Spleen lymphocytes isolated from the mice fed 10t,12c-CLA produced more immunoglobulin (Ig)A and IgM but not IgG when stimulated with concanavalin A (ConA) compared with controls. IgA production from unstimulated spleen lymphocytes was greater in the 10t, 12c-CLA group than in controls. Conversely, 9c,11t-CLA did not affect the production of any of the Ig subclasses. Lymphocytes isolated from 9c,11t-CLA fed mice produced more tumor necrosis factor-alpha than the control group. The proportion of B cells in the spleen lymphocyte population was significantly lower in the 9c,11t-CLA group, and higher in the 10t,12c-CLA group than in the controls. Compared with the control group, the percentage of CD4(+) T cells was lower in the 10t,12c-CLA group, and the percentage of CD8(+) T cells was higher in the 9c,11t-CLA group. Furthermore, the percentage of CD8(+) T cells was higher in the 1:1 mixture group than in controls. The CD4(+)/CD8(+) ratio was lower in the 1:1 mixture group than in controls. These results suggest that 9c,11t and 10t,12c-CLA can stimulate different immunological effects and that the simultaneous intake of the two isomers can change the T cell population.

We have recently shown that alpha-eleostearic acid (alpha-ESA), a conjugated linolenic acid, has a stronger antitumor effect than conjugated linoleic acid (CLA), both in vitro and in vivo. In this study, the oxidative stability of alpha-ESA was examined compared with linoleic acid (LA), alpha-linolenic acid (LnA), and CLA. Thin layers of the FA (LA, 9Z,11 E-CLA, 10E,12Z-CLA, LnA, and alpha-ESA) were auto-oxidized at 37 degrees C, and the FA remaining, the absorbed oxygen volume, the lipid hydroperoxide content, and the TBARS content were determined. The oxidation rate of alpha-ESA was faster than that of the unconjugated FA and CLA (9Z, 11 E-CLA and 10E, 12Z-CLA). However, the lipid hydroperoxide and TBARS contents following alpha-ESA oxidation were low, suggesting production of only small amounts of rapid-reacting secondary oxidation products. Furthermore, the oxidative stability of conjugated FA (CLA and CLnA) in which the carboxylic acid group was esterified with triacylglycerol was greater than that of the FFA. Addition of an antioxidant (alpha-tocopherol) also increased the stability of the conjugated FA to a level similar to that of the unconjugated FA.

A commercial product of CLA contains almost equal amounts of cis-9,trans-11 (c9,t11)-CLA and trans-10,cis-12 (t10,c12)-CLA. We attempted to enrich the two isomers by a two-step selective esterification using Candida rugosa lipase that acted on c9,t11-CLA more strongly than on t10,c12-CLA. An FFA mixture containing CLA isomers was esterified with an equimolar amount of lauryl alcohol in a mixture of 20% water and the lipase. When the esterification of total FA reached 50%, two isomers were fractionated in a good yield: t10,c12-CLA was enriched in FFA, and c9,t11-CLA was recovered in lauryl esters. The FFA were esterified again to enrich t10,c12-CLA. At 27.3% esterification of total FA, the t10,c12-CLA content in FFA increased to 64.8 wt% with 89.3% recovery: The ratio of the content of t10,c12-CLA to that of two isomers was 95.9%. Lauryl esters obtained by the single esterification were employed for enrichment of c9,t11-CLA. After the esters were hydrolyzed, the resulting FFA were esterified again with lauryl alcohol. At 62.0% esterification of total FA, the c9,t11-CLA content in lauryl esters increased to 73.3 wt% with 79.4% recovery: The ratio of the content of c9,t11-CLA to that of two isomers was 95.6%. In a 600-g-scale purification, molecular distillation was effective in separating the reaction mixture into lauryl alcohol, FFA, and lauryl ester fractions.CLA is a group of C18 FA containing a pair of conjugated double bonds in either the cis or trans configuration. It is industrially produced by alkali conjugation of safflower or sunflower oil in propylene glycol or ethylene glycol and contains almost equal amounts of cis-9,trans-11 (c9,t11)-CLA and trans-10,cis-12 (t10,c12)-CLA. The mixture of CLA isomers has various physiological activities, such as reduction of the incidence of cancer (1-3), decrease in body fat content (4,5), beneficial effects on atherosclerosis (6,7), and improvement of immune function (8). Natural CLA is present in meat and milk of ruminant animals (9), and the predominant isomer is c9,t11-CLA, which constitutes over 75% of total CLA (10,11). Although most of the physiological activities have been investigated using a mixture of CLA isomers, it was recently reported that t10,c12-CLA participates in the decrease of body fat (12-14) and that c9,t11-CLA possesses anticancer activity (15). These studies called a great deal of attention to the fractionation of CLA isomers.Recently, the enzymatic method was found to be very effective for the fractionation of CLA isomers. Haas et al. (16) reported that Geotrichum candidum lipase recognized c9,t11-CLA more readily than t10,c12-CLA. They successfully enriched c9,t11-CLA in methyl esters from the products in the early stage of the reaction in which a mixture of CLA isomers was esterified with methanol in an organic solvent system. In addition, c9,t11-CLA was enriched in FFA from the products obtained by hydrolyzing methyl esters of CLA isomers. The purity of CLA isomers can be increased efficiently by their procedure, but the recovery is not g...

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CLA has various physiological activities, and a FFA mixture containing almost equal amounts of cis-9,trans-11 and trans-10,cis-12 CLA (named FFA-CLA) has been commercialized. We attempted to produce MAG of CLA by a two-step successive reaction. The first step was esterification of FFA-CLA with glycerol. A mixture of FFA-CLA/glycerol (1:5, mol/mol), 2 wt% water, and 200 units/g of Penicillium camembertii monoand diacylglycerol lipase was agitated at 30°C to form a homogeneous emulsion. The esterification degree reached 84% after 10 h. To further increase the degree, the reaction was continued with dehydration at 5 mm Hg. The esterification degree reached 95% after 24 h (34 h in total), and the reaction mixture contained 50 wt% MAG and 44 wt% DAG. The second step was glycerolysis of the resulting DAG. The reaction mixture in the first-step esterification was transferred from the reactor to a beaker and was solidified by vigorous agitation on ice. When the solidified mixture was allowed to stand at 5°C for 15 d, glycerolysis of DAG proceeded successfully, and MAG content in the reaction mixture increased to 88.6 wt%. Hydrolysis of the acylglycerols was not observed during the second reaction. FA composition in the synthesized MAG was completely the same as that in the original FFA-CLA, showing that Penicillium lipase does not have selectivity toward FA in the FFA-CLA preparation.Paper no. J10248 in JAOCS 79, 891-896 (September 2002).KEY WORDS: CLA, esterification, glycerolysis, MAG, monoand diacylglycerol lipase, Penicillium camembertii.MAG are very good emulsifiers and are widely used as food additives. MAG are produced industrially by chemical alcoholysis of oils and fats with two molar equivalents of glycerol in the presence of metal catalysts at high temperatures of 210-240°C (1,2). But the process cannot be applied to synthesize MAG of unstable FA. Hence, many research groups have engaged in the synthesis of MAG through lipasecatalyzed reactions (hydrolysis, esterification, glycerolysis, and ethanolysis) (3-8). These reactions were, however, conducted in organic solvent systems, which are not suitable for industrial production of MAG esterified with functional FA. Meanwhile, several organic solvent-free systems also have been reported: (i) glycerolysis with Pseudomonas lipase (9,10); (ii) ethanolysis of TAG with immobilized Candida antarctica lipase (11); and (iii) esterification of FFA with glycerol using Penicillium camembertii mono-and diacylglycerol lipase (referred to as Penicillium lipase) (12). CLA is produced by alkali conjugation of safflower or sunflower oil in propylene glycol or ethylene glycol. The first product is a FFA mixture containing almost equal amounts of cis-9,trans-11 (c9,t11)-CLA and trans-10,cis-12 (t10,c12)-CLA, and the acylglycerols (main component, TAG) esterified with the FFA are also commercially available. The FFA mixture containing CLA isomers has various physiological activities that have been demonstrated in animal models, such as reduction of the incidence of cancer (13-15), d...