The concentration of conjugated linoleic acid isomers and their metabolites in the abdominal fat of rats

The influence of adding Se-enriched yeast (Se-yeast) to a diet containing 2% of a mixture of conjugated linoleic acid isomers (CLAmix) on the profile of CLA isomers and their metabolites (CFA) in abdominal fat of rats was investigated. The results demonstrated that the concentration of CLA isomers and CFA was higher in the abdominal fat of rats fed the diet enriched in both Se-yeast and CLAmix compared with rats fed the diet supplemented only with the CLAmix.


INTRODUCTION
Conjugated linoleic acid (CLA) isomers are present in the fat associated with meat and milk of ruminants (Wahle et al., 2004).Anticarcinogenic, antiatherosclerotic, antidiabetogenic, and antiadipogenic properties have been attributed to CLA isomers, particularly their longer chain metabolites (CFA), and to Se (Belury, 2002).It is likely that the physiological activities of CLA isomer metabolites may overlap with the properties of the parent CLA isomers.Se and CLA isomers also affect bone formation, the immune system, fatty acid and lipid metabolisms, and gene expression in numerous tissues (Banni et al., 2004;Suzuki, 2005).It has also been found that in rats, the concentration of polyunsaturated fatty acids positively correlated with the level of Se in diets (Czauderna et al., 2004).The main aim of this study was to evaluate the influence of a diet enriched in high-selenized yeast (Se) and/or mixture of CLA isomers on the profile of CLA isomers and their metabolites (CFA) in abdominal fat of rats.Silver-ion (Ag + -HPLC), reversed-phase (RP-HPLC) high-performance liquid chromatography with photodiode array detection (DAD) and capillary gas-liquid chromatography (GLC) were adapted to analyse CLA isomers and CFA in rat fat.

MATERIAL AND METHODS
Four groups of female rats (Wistar, Ifz: BOA), 8 weeks of age with an initial body weight of ~200 g, were housed individually as described previously (Korniluk et al., 2006).During a one-week preliminary period the rats were fed a standard Labofeed diet at a submaintenance level (daily ~9 g of the diet per rat) to reduce their body fat.In the experimental period lasting 4 weeks, rats were fed ad libitum with the Labofeed diet enriched in 1.2 ppm Se (as Se-yeast) and/or the CLA isomer mixture (CLAmix) at a level of 2% (Table 1).At the end of the experimental period the rats were killed by CO 2 ; abdominal fat samples were taken and analysed for CLA isomers and CFA concentration.
All of the reagents used, the methods of hydrolysis, derivatization and Ag + -HPLC or GLC configuration were as previously described (Korniluk et al., 2006).The derivatized CLA isomers and CFA were determined by PR-HPLC (Czauderna et al., 2002), while non-derivatized fatty acids were analysed using Ag + -HPLC (Czauderna et al., 2003) and RP-HPLC (Czauderna et al., 2002).The CLAmix was supplied by Larodan Fine Chemicals AB (Sweden).Eighty-three per cent of the total Se content of selenized yeast (Se-yeast) represent Se in the form of Se-methionine incorporated into the proteins of Saccharomyces cerevisiae.Seenriched yeast was donated by Sel-Plex (Alltech Inc., USA).
Statistical analyses of the effects of Se-yeast on the concentration of CLA isomer(s) and CFA in fat were conducted using the nonparametric Mann-Whitney U test.Differences were considered significant at the P<0.05 or P<0.01 level.

RESULTS AND DISCUSSION
The concentrations of CLA isomers and their metabolites containing a pair of conjugated double bonds (CFA) was under the detection limit in rats fed the control and only Se-enriched diets (Table 1).No lesions or symptoms of Seyeast or CLA isomer intoxication were found in rats fed diets enriched in CLA isomers and/or Se-yeast.The diet enriched in Se-yeast, regardless of the presence of CLAmix, had a very small influence on the body weight gain of rats, while the dietary CLAmix tended to decrease the weight of rats (i.e. an antiobesity property of CLA isomers).As expected, both diets enriched in the CLAmix resulted in a significant increase in the concentration of CLA isomers in abdominal fat.Moreover, addition of Se-yeast to the diet with CLAmix elevated the accumulation of CLA isomers, particularly c9t11CLA and c,cCLA, in the abdominal fat of rats compared with the group fed the diet with only CLAmix (Table 1).Ag + -HPLC investigations demonstrated that two unidentified non-CLA fatty acids (CFA) in abdominal fat have an absorbance maximum at ~234 nm due to the presence of CLA ISOMERS AND METABOLITES IN ABDOMINAL FAT OF RATS conjugated double bonds and had retention times of 18.3±0.2and 19.3±0.2 min.Moreover, detailed RP-HPLC studies using direct DAD-detection of free fatty acids as well as DAD-monitoring after derivatization of fatty acids of abdominal fat demonstrated that the two unidentified non-CLA fatty acids contained a pair of conjugated double bonds and had an additional third non-conjugated bond.These fatty acids (CFA) contained three double bonds and twenty-carbon atoms in a chain because they eluted faster than CLA isomers, and slower than aracidonic acid.Thus, based on our earlier works (Czauderna et al., 2004;Korniluk et al., 2006), and studies of Banni et al. (2004), as well retention times of detected CFA and their UV spectra, we suggest that these fatty acids are higher metabolites such as c8t12c14C20:3 and c8c11t13C20:3.Based on current chromatographic analyses and our previous studies (Czauderna et al., 2004;Korniluk et al., 2006), we suggest that CLA isomers could be metabolized in vivo into long-chain conjugated PUFA (via ∆6-, ∆5-desaturation and elongation of CLA isomers) using the same pathway as linoleic acid (Czauderna et al., 2004).Our results demonstrate that the concentration of CFA is higher in the abdominal fat of rats fed the diet 13508 a 33737 a 60141 a 1 means in columns with the same letter are significantly different at a,b P<0.05; 2 t,t -trans,trans CLA isomers; c,c -cis,cis CLA isomers; Σ -all isomers -sum of all CLA isomers and CFA (metabolites of CLA isomers); 3 the weight gain of rats after 4 weeks feeding with experimental diets; 4 the profile of CLA isomer mixture: 0.06% -the sum of t11t13, t10t12, t9t11 and t8t10; c10t12 -49.2%; c9t11 -48.0%; 1.026% -the sum of c11c13, c10,c12, c9c11 and c8c10; 5 NQ: below the quantification limit (L Q ) enriched in Se-yeast and CLAmix compared with rats fed the diet with only CLAmix (Table 1).The present data are consistent with our previous results showing that dietary selenate stimulates the formation of higher metabolites in femoral muscles and liver of rats fed diets with CLA isomers (Czauderna et al., 2004).These results clearly indicate that dietary t,tCLA isomers are discriminately accumulated into abdominal fat, in contrast with being preferentially incorporated into phospholipids and triacylglycerols of rat liver (Czauderna et al., 2004).In agreement with our previous studies, addition of Se-yeast to the diet with CLAmix slightly increased the accumulation of t,t isomers in fat.
The concentration ratio of c9t11CLA to t10c12CLA in fat of rats fed the diets enriched in CLAmix with or without Se-yeast was higher compared with the concentration ratio of these isomers in CLAmix added to the diets (i.e.1.49-1.53vs 0.98; Table 1).These results are in agreement with our previous studies (Czauderna et al., 2004;Korniluk et al., 2006) and with that of Belury (2002), in which t10c12CLA and t10t12 CLA were also driven through β-oxidation in rat adipose cells, muscles, kidneys and liver more efficiently than their 9,11 homologues.

CONCLUSIONS
We hypothesize that dietary Se-yeast decreases the catabolism of CLA isomers as well as stimulates the capacity of ∆6 desaturase and elongase in rats.Finding that the addition of Se-enriched yeast to a diet enriched in CLAmix increased the concentration of CLA isomers and, in particular, of their higher metabolites (CFA) in fat is valuable information for nutritionists trying improve the nutritive value of food.

Table 1 .
Mean concentrations of CLA isomer(s) (µg/g) 1 in abdominal fat of rats fed a diet enriched to 1.2 ppm Se (as Se-yeast) and/or 2% of the CLA isomer mixture