The influence of dietary conjugated linoleic acid isomers and selenized yeast on the fatty acid profile of the spleen , pancreas and kidneys of rats

The influence of 1.2 ppm dietary selenium added in the form of selenized yeast (Se-yeast) and of a mixture of conjugated linoleic acid (CLA) isomers added to a level of 1 or 2%, of cis9trans11CLA (c9t11) added to 1%, and of trans10cis12CLA (t10c12) added to 1% on the body and organ gains of rats and on the concentration of CLA isomers and other fatty acids in the spleen, pancreas and kidneys was investigated. Total fatty acids in the studied organs were separated as methyl esters using long-capillary gas-liquid chromatography with flame-ionization detection, while nonmethylated fatty acids containing conjugated double bonds were examined using silver-ion liquid chromatography with fotodiode detection. Free fatty acids were obtained by mild saponification, whereas their methyl esters, by baseand acid-catalyzed methylation. Feeding t10c12 and Se-yeast resulted in the highest increase in rat growth and organ gain. All diets enriched in CLA isomer(s) significantly elevated the concentration of CLA isomers in the organs; the highest concentration of CLA isomers was found in the pancreas, the smallest, in the spleen. The addition of Se to diets containing CLA isomer(s) usually showed a statistical tendency or increase in the concentration of CLA isomers in the organs. Our results demonstrate that t10c12 was metabolized faster than c9t11 in the pancreas and kidneys, whereas the addition of Se-yeast to the diets with CLA isomer(s) reduced the rate of t10c12 metabolism. A 2% dietary content of a mixture of CLA isomers or of t10c12 usually tended to increase or significantly increased the concentration of linoleic, linolenic, and arachidonic acids and of n-3 long-chain polyunsaturated fatty acids (n-3 PUFA) in the organs. Dietary c9t11 and a 1% content of the mixture of CLA isomers weakly influenced the concentrations of these fatty acids in the organs, whereas the diet enriched simultaneously in c9t11 and Se-yeast resulted in a statistical tendency or increase in the concentration of long-chain n-3 PUFA and the 1 Corresponding author: e-mail: m.czauderna@ifzz.pan.pl Journal of Animal and Feed Sciences, 16, 2007, 121–139 122 FATTY ACID AND CLA IN ORGANS OF RATS sum of PUFA in the organs compared with the control group and rats fed the diet containing only c9t11. The obtained results suggest that t10c12 and, in particular, 2% of the mixture of CLA isomers decreased the capacities of Δ9-, Δ6-, Δ5and Δ4-desaturases, as well as of cyclooxygenase-1, and/or the inducible form, cyclooxygenase-2, at the level of mRNA, protein, or activity.


INTRODUCTION
The term conjugated linoleic acid (CLA) isomers refers to a collection of positional and geometric isomers of octadecadienoic acid with conjugated double bonds.The recent increased interest in CLA isomers results from numerous animal investigations associating conjugated fatty acids, particularly cis9trans11CLA (c9t11), with healthpromoting effects (Belury, 2002;Naumanna et al., 2006).Furthermore, trace elements, including selenium (Se), are also crucial nutrients for humans as well as all animals.Se is a component of a number of important selenoproteins and enzymes required for such functions as antioxidant and anticancer defence, reduction of inflammation, thyroid hormone production, DNA synthesis, fertility and reproduction (Kornitzer et al., 2004;Rayman, 2004).Consequently, adequate dietary intake of CLA isomers and Se is essential for good health.Interestingly, numerous animal investigations found that the contents of mono-(MUFA) and polyunsaturated fatty acids (PUFA), especially in serum cholesterol esters and phospholipids, were positively correlated with Se concentrations in the diet (Crespo et al., 1995).According to Tapiero et al. (2003) and Suzuki (2005), half of known Se-proteins have been implicated in antioxidant functions, and Se-cysteine (Se-Cys) is essential in the active centres of Se-enzymes that carry out redox reactions, e.g., glutathione peroxidase (GPx), thyroid hormone deiodinase families, or thioredoxin reductase (Tapiero et al., 2003).Phospholipid hydroperoxide cGPx in particular interacted more directly than cytosolic and mitochondrial cGPx in protecting PUFA from peroxidation damage (Crespo et al., 1995).Recent investigations have shown that feeding diets enriched in high-selenized yeast (Se-yeast; 83-60% Se as Se-methionine (Se-Met) (Rayman, 2004)) to animals results in effective induction of antioxidant enzymes, indicating that the animal converts this chemical form of Se-Met to Se-Cys, which is an essential amino acid for the synthesis of this Se-protein (Schomburga et al., 2004;Whanger, 2004;Korniluk et al., 2006).Thus, these protective effects of dietary Se (as high-selenized yeast) on the deposition of CLA isomers and other fatty acids in the body of mammals may also be plausible.
In recent studies we found that dietary Se (as high-selenized yeast and selenate) and CLA isomers influence the amount of CLA isomers and other fatty acids in the liver and femoral muscles of rats (Czauderna et al., 2004a,b;Korniluk et al., 2006).Considering the above, we hypothesized that addition of high-selenized yeast (Se-yeast) to rats' diets containing CLA isomers caused changes in the yield of the accumulation of CLA isomers and other fatty acids in the examined organs of rats.Therefore, the objective of the present study was to investigate the effect of adding Se-yeast to diets enriched to 1 or 2% of a mixture of CLA isomers or to 1% of individual CLA isomers (i.e.c9t11 and t10c12) on the concentration of fatty acids, particularly CLA isomers, in the spleen, pancreas, and kidneys of rats.Moreover, the influence of the experimental factors on the body weight gain of rats, the weight of these organs and feed intake was also determined.

Animals and treatments
Ten groups of 8-week-old female rats (Wistar, Ifz: BOA) were housed individually in plastic cages as described by Korniluk et al. (2006).The animals were housed and handled in accordance with protocols approved by the Local Animal Care and Use Committee (The Agricultural University of Warsaw, Poland).During a one-week preliminary period the animals were fed a standard Labofeed H diet (Pastuszewska et al., 2000) given at a submaintenance level (~9 g of the diet per rat daily) to reduce their body fat.In the experimental period lasting 4 weeks, the rats were fed ad libitum with the Labofeed H diet enriched to 1 or 2% of a mixture of CLA isomers, 1% cis9trans11CLA (c9t11), 1% trans10cis12CLA (t10c12) and/or 1.2 ppm Se, as Se-yeast (Rayman, 2004).After 28 days of feeding the experimental diets the rats were euthanized by CO 2 and the spleen, pancreas and kidneys were removed, weighed, and analysed for their fatty acids.

Chemicals
Water and organic solvents were HPLC grade, whereas all of the remaining reagents were analytical grade.The CLA isomer mixture, individual CLA isomers (i.e.c9t11 and t10c12) were supplied by Larodan Fine Chemicals AB (Sweden).The purity and composition of the CLA isomer mixture and individual isomers were examined by Ag + -HPLC and GLC (Table 1) (Czauderna et al., 2003a(Czauderna et al., , 2005)).All fatty acid standards were provided by Sigma (USA), while n-heptane (95%) was purchased from Lab-Scan (Ireland).A non-commercial Se-yeast sample was donated by Sel-Plex, Alltech Inc. (USA).The compositions of the Labofeed H diet and dietary Se-yeast sample are presented in Table 2.  (Czauderna et al., 2003a(Czauderna et al., , 2005) ) Table 2.Chemical composition (g/100 g diet) and the mean energy value of the Labofeed H diet (Pastuszewska et al., 2000) and Se-yeast (Sel-Plex) (Rayman, 2004) Item

Saponification and fatty acid extraction
Spleen, pancreas and kidneys samples were frozen, lyophilized and the obtained residues were stored at -20 o C until saponification.Finely powdered organ samples (∼50 mg) were placed in vials and treated with 2 ml of 2M KOH in water, 2 ml of 1M KOH in methanol, and 50 µl of an internal standard (nonadecanoic acid in chloroform: 17 mg/ml) were added.The resulting mixtures were flushed with a stream of argon (Ar) for ~5 min.Finally, the obtained solutions in tightly closed tubes were vigorously mixed and heated at 92-95 o C for 10 min, next cooled for 10 min at room temperature and sonicated for ~10 min.The obtained solutions were protected from the light and stored for overnight in sealed vials at room temperature under Ar.Then, 3 ml of water were added to the hydrolysates and solutions were again vigorously vortexed.The obtained solutions were acidified with 4M HCl to pH 1-2 and free fatty acids (FAs) were extracted 4 times, each time using 3 ml of dichloromethane (DCM).The lower DCM layer was dried with ∼100 mg of Na 2 SO 4 .To avoid loss of free FAs, extraction was repeated 4 times, each time using 3 ml of n-heptane.Afterward, the upper n-heptane layer was combined with the DCM layer and the organic solvents were removed under a stream of Ar.The obtained residues were stored at -20 o C until methylation.

Preparation of fatty acid methyl esters (FA-MEs)
To residues, 2 ml of 2M NaOH in methanol and 50 µl of BHT solution in methanol (20 mg/ml) were added while mixing, then flushed with Ar, and reacted for 1 h at 40 o C.After cooling, 2 ml of 25% BF 3 in methanol were added to the reaction mixture, flushed with Ar and heated for 1 h at 40 o C. To the cooled reaction mixtures 5 ml of water were added and then FA-MEs were extracted with 5 ml of n-hexane.The supernatant was transferred to a vial.Separation of FA-MEs was carried out by gas liquid-chromatography (GLC), while FA-MEs containing conjugated double bonds were also re-analysed using silver-liquid chromatography (Ag + -HPLC) with photodiode array detection (DAD) at 234 nm (Czauderna et al., 2003a).

Analytical conditions
The analyses of FA-MEs were performed on an Agilent 6890N GC equipped with a CP7489 fused silica capillary column (100 m × 0.25 mm i.d.× 0.2 μm film thickness; Varian, USA) and FID, while split injections were performed using an Agilent 7683 autosampler (Czauderna et al., 2005); the Ag + -HPLC equipment was as previously described (Czauderna et al., 2003a).The concentrations of all fatty acids were calculated from lyophilized spleen, pancreas and kidneys samples (i.e.dry matter).

Statistical analyses
The data are presented as means.Statistical analyses of the effects of Se or the CLA isomers in the diets were conducted using the nonparametric Mann-Whitney U test for comparing independent experimental groups, while statistical analyses of interactions between the CLA isomer(s) and Se-yeast were performed using two-factorial ANOVA analysis (CLA isomer(s) x Se).Statistica ver.6 and Microsoft Office Excel software were used in the statistical analyses (Statistica, 2002).Differences were considered significant at P<0.05.

The influence of dietary Se-yeast and CLA isomers on relative body weight gain, feed intake and weight of organs
The influence of diets enriched in CLA isomer(s) and/or Se-yeast on the feed intake and the growth of rats is summarized in Table 1.As can be seen from these results, the diet enriched in the t10c12 isomer and Se-yeast most efficiently elevated the body weight gain (BWG) of rats and the weight of the pancreas and kidneys of rats compared with animals fed the diet supplemented with only Se-yeast as well as with the control rats.Moreover, the diet with t10c12 and Se-yeast most effectively improved feed conversion efficiency (FCE) and tended to cause a smaller decrease in the weight of the spleen compared with the control rats (Table 1).Unexpectedly, the opposite phenomenon was observed when rats were fed the diet containing 2% of the CLA isomer mixture and when animals were fed the diet enriched in only Seyeast or one containing both the c9t11 isomer and Se-yeast.The diet to which both c9t11 and Se-yeast were added effectively decreased the BWG of rats and showed a tendency to lower FCE, whereas the diet enriched in only 2% of the CLA isomer mixture decreased feed intake as well as tended to most effectively decrease BWG.Feeding the diet containing 2% of the CLA isomer mixture also resulted in a lower pancreas weight compared with the control and other experimental groups.

The influence of dietary CLA isomer(s) and Se-yeast on the accumulation of CLA isomers in spleen, pancreas and kidneys
Detailed chromatographic investigations showed that feeding CLA isomer(s) resulted in the efficient accumulation of CLA isomer(s) in the spleen, pancreas and kidneys of rats (Tables 3-8).In addition, increasing the dietary concentration of the CLA isomer mixture induced a significant rise in the concentration of CLA isomers (P<0.01) in all assayed organs.The accumulation of CLA isomer(s) in pancreas was higher than in the spleen and kidneys of rats fed diets supplemented with CLA isomer(s), regardless of the presence of Se-yeast.Interestingly, the accumulation of two isomers (i.e.c9t11 and t10c12) in the pancreas of rats fed the diets enriched to 1 or 2% of the CLA isomer mixture reduced the concentrations of other assayed fatty acids in the pancreas (Tables 4 and 7) in comparison with the concentration of these fatty acids in this organ in control rats.3 4 below the quantification limit (3 × the detection limit) 5 value of (c9t11)/(t10c12)ratio: 2.4530 and 1.6202 for the 1% CLA and 1% CLA+Se groups, respectively A higher concentration of t10c12 compared with that of c9t11 was observed in spleen of rats fed the diet containing 2% of the CLA isomer mixture regardless of the addition of Se-yeast.On the other hand, the concentration of c9t11 was substantially higher than the concentration of t10c12 in the pancreas and kidneys of animals fed the diets containing the CLA isomer mixture without/with Se-yeast.
Addition of Se-yeast to the diets enriched in CLA isomer(s) increased the yield of CLA isomer(s) accumulation in the spleen and pancreas in comparison with the amount of these CLA isomer(s) in spleen and pancreas of rats fed the diet enriched in only CLA isomer(s).
The addition of Se-yeast to the diets containing 1% CLA isomers stimulated the accumulation of c9t11 and the sum of both isomers, while tended to decrease the concentration of t10c12 in kidneys in comparison with the amount of these isomers in kidneys of rats fed the diet enriched in only 1% the mixture of CLA isomers or t10c12, respectively.Surprisingly, the presence of Se-yeast in the diet enriched to 2% of the mixture of CLA isomers reduced the accumulation of CLA isomer(s) in kidneys compared with animals fed the diet enriched in only 2% CLA isomers.The current study also investigated the relationship between the percentage content of c9t11 and t10c12 isomers in all analysed organs of rats fed the diets enriched with a mixture of c9t11 and t10c12 isomers.The obtained results demonstrate that the accumulation of CLA isomers is selective and clearly show that c9t11 is preferentially accumulated in the pancreas and kidneys (Tables 4, 5, 7 and  8).Therefore, the ratio of (c9t11)/(t10c12) in the dietary CLA isomer mixture (Table 1) was lower than the ratio of these isomers in the pancreas and kidneys of rats fed the diet enriched in the CLA isomer mixture (Tables 4, 5, 7 and 8).On the other hand, the concentration ratio of (c9t11)/(t10c12) in the spleen of rats fed the diets enriched with the CLA isomer mixture (Tables 3 and 6) were lower in comparison with the values of this ratio in pancreas and kidneys, and similar to the concentration ratio of (c9t11)/(t10c12) in the dietary mixture of CLA isomers (see Table 1).
As can be seen from results presented in Tables 3-8, addition of Se-yeast to the diets containing the mixture of CLA isomers usually resulted in a relative increase of the percentage of t10c12 in the spleen, pancreas and kidneys compared with these organs of rats fed the diets containing only the mixture of CLA isomers.A,B -P<0.01;a,b -P<0.05; 3 abbreviations for FA(s) and other items see Table 3; 4 below the quantification limit (3 × the detection limit); 5 value of (c9t11)/(t10c12)ratio: 1.430 and 1.449 for the 2% CLA and 2% CLA+Se groups, respectively

Influence of experimental diets on the non-conjugated fatty acid constituent in rat organs
Feeding 1% of a mixture of CLA isomers or t10c12 isomer to animals usually showed a tendency to elevate or significantly elevated the concentration of C18:0 as well as SFA in all assayed organs of rats; this effect was strongest in the spleen and weakest in the kidneys (Tables 3-5).As expected, the stimulation of accumulation of these fatty acids was usually increased in all assayed organs if the diet was enriched to a higher concentration of the CLA isomer mixture (Tables 6-8).Moreover, the addition of Se-yeast to the diets enriched in the CLA isomer mixture or t10c12 usually resulted in a further increase in the SFA and C18:0 concentrations in all rat organs.Consequently, the value of the Δ9-desaturase index (Δ9index) in all analysed organs of rats fed the diets enriched in the mixture of CLA isomers and t10c12 decreased compared with that in the organs of the control rats (Tables 3-8).The reduction of Δ9index values was stronger if the diet was enriched in 2% the mixture of CLA isomers (Tables 3-5 vs 6-8).In addition, this effect of the CLA isomer mixture and t10c12 was usually stronger in all organs if the diets with these isomer(s) were also enriched in Se-yeast.The experimental diet enriched only in Seyeast also caused a decrease in the Δ9index in all organs, probably due to inhibited steaoryl-CoA desaturase mRNA expression (Tables 3-5).
On the other hand, dietary c9t11 showed a tendency to decrease or statistically decreased the concentration of SFA as well as C18:0 in all assayed organs of rats compared with the control organs, whereas addition of Se-yeast to the diet containing c9t11 resulted in a higher concentration of these fatty acids compared with control organs.
Our current results as well as our previous studies (Niedźwiedzka et al., 2006) have clearly shown that dietary CLA isomer(s), regardless of the addition of Se-yeast to diets, usually showed a tendency to increase or statistically increased the concentration of linoleic acid (LA) in all examined organs (Tables 3-8).The stimulating influence of the CLA isomer mixture on the accumulation of LA in these organs is stronger if the diet is enriched in a higher concentration of CLA isomers.These diets, regardless of the presence of Se-yeast, also usually tended to increase or significantly elevated the concentration of arachidonic acid (C20:4n-6) in all assayed organs.As expected, an increase in the concentration of C20:4n-6 in all organs was observed with the higher concentration of the CLA isomer mixture in the diet.
The diet containing only Se-yeast usually caused a minute influence on the concentration of α-LNA as well as other long-chain PUFA in the organs, whereas all diets enriched in CLA isomer(s) showed mostly a tendency or statistically significant increase in the accumulation of α-linolenic acid (α-LNA) in the examined organs.Moreover, the diets enriched in t10c12 and 2% of the CLA isomer mixture, particularly those also containing Se-yeast, usually tended to increase or significantly increased the concentration of long-chain n-3 PUFA (i.e.C20:5n-3, C22:5n-3 and C22:6n-3) and the sum of polyunsaturated fatty acids (PUFA) in the organs of rats.In contrast, dietary c9t11 and the lower concentration of the administered CLA isomer mixture weakly influenced the concentrations of these fatty acids in organs, whereas the diet enriched simultaneously in c9t11 and Se-yeast resulted in a tendency towards or statistically significant increase in the concentration of n-3 PUFA and PUFA in organs compared with the control group and rats fed the diet containing only c9t11.

Impact of dietary Se-yeast and CLA isomers on rat growth
No macroscopic lesions or toxic symptoms caused by dietary Se-yeast and CLA isomers were observed in animals fed experimental diets.In fact, the diet containing 2 ppm of Se would not be toxic for rodents (like rats or mice) because only chronic ingestion of inorganic Se compounds, selenite in particular, at a rate of more than 5 ppm can be hepatotoxic and teratogenic in humans and animals (Tapiero et al., 2003;Tinggi, 2003).The LD 50 for Se is about 5 mg Se/kg of body mass for rats, thus, this corresponds with a diet containing ~50 ppm Se (i.e.20 g of the Labofeed H diet enriched to 50 ppm Se per rat per day).Furthermore, in contrast to tRNA Se-cysteine , tRNA Met does not discriminate between Se-Met and methionine (Met).Therefore, Se-Met is incorporated into body proteins in place of methionine (Met) (Tapiero et al., 2003).Therefore, a greater percentage of dietary Se-Met (from Se-yeast) is accumulated non-specifically into body proteins in place of Met, especially in low-Met diets.Se-Met proteins are a safe-storage form of Se in the body of laboratory animals and humans.
The current results are consistent with our earlier studies, which have also indicated that the diet enriched in the t10c12 isomer and inorganic Se (as selenate) is the most potent in terms of elevating the body weight of rats as well as results in the best FCE (Czauderna et al., 2003b(Czauderna et al., , 2004a)).Moreover, our results are consistent with other earlier investigations showing that dietary CLA isomer(s) is the most potent factors in terms of antiobesity properties (Alasnier et al., 2002;Belury, 2002;Czauderna et al., 2004b).The levels of adrenaline and noradrenaline were, in fact, significantly higher in experimental animals gavaged with the CLA isomer(s).Based on our previous studies and current results we suggest that the interaction between c9t11 and t10c12, especially at the higher concentration of the CLA mixture, most efficiently enhances sympathetic nervous activity that leads to increased energy metabolism and eventual reduction of adipose tissue (Belury, 2002;Czauderna et al., 2003b).

The influence of dietary Se-yeast and CLA isomer(s) on the accumulation of CLA isomers in rat organs
The obtained results are consistent with our previous observations in which dietary inorganic selenium (as selenate) also increased the concentration of CLA isomers as well as other PUFA in muscles and organs of rats fed a diet enriched simultaneously in selenate and CLA isomer(s) (Czauderna et al., 2004a,b).Considering the above results, we could argue that dietary organic (as Se-yeast) and inorganic selenium protects against peroxidation of unsaturated fatty acids and, like tocopherols, provides a line of defence against reactive species, which can damage membranes and other cell structures.
The present results clearly shows that the yield CLA isomer accumulation depended upon the rat organ and geometrical configuration of the CLA isomers added to the diet.So, our current results are in agreement with the observations of Alasnier et al. (2002) and our previous studies on rats (Czauderna et al., 2003b(Czauderna et al., , 2004a,b;,b;Niedźwiedzka et al., 2006) showing that in the cells of the pancreas, kidneys, brain, liver, muscles and adipose tissues, t10c12 and t10t12CLA isomers are more efficiently driven through β-oxidation than their 9,11 homologues.In contrast, the amount of c9t11 and t10c12 in the spleen of rats fed the diets enriched in the CLA isomer mixture were similar.So, the current results are in agreement with our previous observations in which the amounts of these two CLA isomers in the spleen of rats fed diets enriched in the isomer mixture, irrespective of the presence of selenate, was also similar to their amount in dietary CLA isomer mixtures (Niedźwiedzka et al., 2006).The presented results suggest that in rat spleen, the rates of metabolism of dietary c9t11 and t10c12 are similar.However, it is also possible that the yield of c9t11 accumulation is lower in comparison with t10c12.This second suggestion is fully supported by the results summarized in Table 3 and our previous studies (Niedźwiedzka et al., 2006); indeed, the concentration of c9t11 was lower than the concentration of t10c12 in spleen of rats fed diets enriched in the same concentration of the individual isomer (i.e.c9t11 and t10c12, respectively; see the groups c9t11 and t10c12).
The present study clearly demonstrated that the dietary Se-yeast stimulated the accumulation of t10c12 in the examined organs of rats.This is in good agreement with our previous studies (Czauderna et al., 2003b(Czauderna et al., , 2004a,b;,b;Niedźwiedzka et al., 2006) in which inorganic (as selenate) and organic Se-compounds (as Seyeast) also increased the percentage of t10c12 at the expense of c9t11.Therefore, we hypothesize that both chemical forms of dietary Se result in decreasing the capacity of β-oxidation of t10c12 and probably also t10c12 isomers in selected tissues and organs of rats fed diets enriched in both the mixture of CLA isomers and Se compared with animals fed feed containing only the mixture of CLA isomers.

Effect of dietary Se-yeast and CLA isomers on the concentration of non-conjugated fatty acids in rat organs
In the current study, designed specifically to investigate the influence of Se-yeast and CLA isomers, we were able to confirm our hypothesis that dietary compounds affect the concentrations of non-CLA fatty acids in all analysed organs of rats.The obtained results are consistent with our previous investigations (Czauderna et al., 2004;Korniluk et al., 2006) and other studies (Alasnier et al., 2002;Kang et al., 2004) which demonstrated that dietary t10c12 as well as the mixture of CLA isomers were responsible for the change in the concentration of SFA and MUFA in organs and tissues of animals fed CLA isomer(s) due to decreasing the Δ9desaturation of FAs such as C14:0, C16:0 or C18:0 (Belury, 2002;Wahle et al., 2004).Indeed, recent studies have demonstrated that CLA isomer(s), particularly t10c12 CLA, reduce the capacity of Δ9-desaturase, inhibit steaoryl-CoA desaturase mRNA expression and fatty acid synthesis (Alasnier et al., 2002;Belury, 2002;Kang et al., 2004).Moreover, the current investigations are consistent with our previous studies (Czauderna et al., 2003b(Czauderna et al., , 2004a,b;,b;Niedźwiedzka et al., 2006), which also suggested that dietary inorganic Se (as selenate) and organic selenium (as Se-yeast) decreased the capacity of Δ9-desaturase in muscles and organs of rats.
It is well-evidenced that diets enriched in CLA isomer(s) result in changes in the concentrations of LA and LNA in the bodies of studied animals (Alasnier et al., 2002;Belury, 2002;Heird and Lapillonne, 2005).Indeed, CLA isomers as well as LA are metabolized via the same enzymatic system such as Δ6 desaturaseelongase-Δ5 desaturase.Consequently, CLA isomers compete for the same enzymes as LA (Belury, 2002;Heird and Lapillonne, 2005) and decrease the rate at which LA and its product (i.e.arachidonic acid) are metabolized.Considering the above, we suggest that the higher accumulation of LA in organs could also be due to its slower metabolism in the presence of dietary CLA isomer(s).There is also increased accumulation of arachidonic acid (C20:4n-6) in organs of rats, which may be produced by dietary CLA isomer(s), irrespective of the presence of Se-yeast (Tables 3-8).In fact, dietary CLA isomer(s) reduce arachidonatederived eicosanoids synthesis through inhibition of the constitutive enzyme, cyclooxygenase-1, and/or the inducible form, cyclooxygenase-2, at the level of mRNA, protein, or activity.CLA isomers or their elongated and desaturated products may also act as substrates or antagonist for cyclooxygenase, thereby decreasing available enzyme for arachidonate (Belury, 2002;Heird and Lapillonne, 2005).
In our study, the diets enriched in CLA isomer(s), particularly those containing Se-yeast, usually tended to or statistically increased the concentration of longchain n-3 PUFA and the sum of polyunsaturated fatty acids (PUFA) in the organs of rats.Indeed, α-LNA is metabolized via the same desaturases and the elongase as LA (Belury, 2002).Other recent investigations (Alasnier et al., 2002;Wahle et al., 2004) also showed that a mixture of CLA isomers, or t10c12 in particular, decreased the formation of higher metabolites of LA and LNA due to decreasing the capacity of Δ6-and Δ5-desaturation of LA and α-LNA in human cells.This influence of CLA isomers and especially t10c12 is in agreement with our current and previous observations on the concentration of other polyunsaturated fatty acids in the organs of rats (Korniluk et al., 2006).
In our trial, feeding the diets enriched in t10c12 and 2% of a CLA isomer mixture, particularly those also containing Se-yeast, usually tended to or significantly increased the concentration of long-chain n-3 PUFA in all analysed organs of rats.The possible mechanism through which the accumulation of these long-chain n-3 PUFA was increased by CLA isomers, particularly t10c12, is by the decrease in the capacity of Δ6-, Δ4-desaturations and elongation of these long-chain n-3 PUFA to very-long chain n-3 PUFA (Heird and Lapillonne, 2005).These dietary CLA isomers also inhibited enzymes involved in the synthesis of eicosanoids from long-chain n-3 PUFA (Heird and Lapillonne, 2005).Another reason raises the possibility that elongated and desaturated metabolites of CLA isomer(s) compete for the same enzymes as long-chain n-3 PUFA.Taken together, it seems reasonable to suggest that dietary CLA isomers, particularly t10c12, decreased the rate of the metabolism of these fatty acids.

CONCLUSIONS
Feeding CLA isomers, irrespective of adding dietary Se-yeast, increased the concentration of these isomers in the organs of rats.In the current investigations, we could confirm our earlier suggestion that dietary Se-yeast, inorganic Se (as selenate) and CLA isomer(s) were responsible for the modification of the MUFA/ SFA ratio.Our results suggest that the mixture of t10c12 and c9t11, particularly t10c12, apparently decreased the capacity of Δ9-, Δ6-, Δ5-and Δ4-desaturation of unsaturated fatty acids in the examined organs of rats.Based on the current results and our previous studies, we suggest that the simultaneous administration of t10c12 and Se-yeast had a beneficial influence on the growth of rats and efficiency of feed utilization.The diets enriched in CLA isomer(s), particularly together with Se-yeast, elevated the concentration of PUFA in organs.The interaction between dietary c9t11 and Se-yeast stimulated the yield of accumulation of n-3 PUFA and PUFA in the organs of rats.
abbreviations for FA(s) and other items see Table3 abbreviations for FA(s) and other items see Table3

Table 3 .
The concentration of fatty acids in spleen of rats fed for 4 weeks with cis9trans11CLA (c9t11), trans10cis12CLA (t10c12) and 1% CLA isomer mixture (CLA) with or without Se-yeast (SeY) Group

Table 6 .
The concentration of fatty acids in the spleen of rats fed for 4 weeks with 2% CLA isomer mixture with or without Se-yeast 2 means in rows with the same letters are significantly different: A,B -P<0.01;a,b-P<0.05; 3abbreviations for FA(s) and other items see Table34 below the quantification limit (3 × the detection limit); 5 value of (c9t11)/(t10c12)ratio: 0.982 and 0.867 for the 2% CLA and 2% CLA+Se groups, respectively

Table 7 .
The concentration of fatty acids in the pancreas of rats fed for 4 weeks with the 2% CLA isomer mixture with or without Se-yeast 1 2 means in rows with the different letters are significantly different: A, B -P<0.01; a,b -P<0.05; 3 abbreviations for FA(s) and other items see Table3; 4 below the quantification limit (3 × the detection limit); 5 value of (c9t11)/(t10c12) ratio: 1.359 and 1.337 for the 2% CLA and 2% CLA+Se groups, respectively

Table 8 .
The concentration of fatty acids in kidneys of rats fed for 4 weeks with the 2% CLA isomer mixture with or without Se-yeast 1 2 means in rows with the different letters are significantly different: