Changes in the fatty acid profile of the subcutaneous fat of swine throughout fattening as affected by dietary conjugated linoleic acid and monounsaturated fatty acids

The fatty acid profile of the subcutaneous fat of pigs and its evolution throughout fattening as affected by dietary conjugated linoleic acid (CLA), monounsaturated fatty acids (MUFA), and their interaction (CLAxMUFA) were studied. Three levels (0, 1, and 2%) of an enriched CLA oil (28% cis-9, trans-11 and 28% trans-10, cis-12 CLA) were combined with two levels of MUFA (low, 19% average; and high, 39% average) for pig feeding (288 gilts). Subcutaneous shot-biopsies were taken from 48 animals at the beginning of the trial (S1, 70 kg), 14 days later (S2, 80 kg), and at slaughter (S3, 107 kg). Inclusion of CLA in the diet caused an increase during fattening in cis-9, trans-11 CLA, trans-10, cis-12 CLA, and saturated fatty acids (SFA) contents of pig backfat and a decrease in MUFA and polyunsaturated fatty acids (PUFA). MUFA supplementation also led to a MUFA enrichment of backfat. The interaction CLAxMUFA affected the SFA content. The rates of accumulation of CLA isomers, SFA, and MUFA throughout the trial did not follow a linear behavior, such rates being higher from S1 to S2 than from S2 to S3. These rates were also influenced by dietary CLA and MUFA levels. The increase in the ratio of saturated to unsaturated fatty acids of backfat caused by dietary CLA might be balanced by supplementation of pig diets with MUFA.     

Conjugated linoleic acid-induced fatty liver can be attenuated by combination with docosahexaenoic acid in C57BL/6N mice

We investigated the effect of dietary combination of conjugated linoleic acid (CLA) and docosahexaenoic acid (DHA) to attenuate CLA-induced fatty liver in C57BL/6N mice. Mice were fed semisynthetic diets that contained either 6% high linoleic safflower oil (HL-SAF), 4% HL-SAF + 2% CLA, or 3.5% HL-SAF + 2% CLA + 0.5% DHA for 4 weeks. This 4 week feeding of CLA showed hepatic lipid accumulation concomitant with the decrease in adipose tissue weight in mice. However, 0.5% supplementation of DHA to the CLA diet could alleviate fatty liver without decreasing the antiobesity effect of CLA. The CLA diet promoted fatty acid synthesis in the liver, but DHA supplementation significantly attenuated the increase in enzyme activity induced by CLA. On the other hand, serum adipocytokines, leptin and adiponectin, were drastically decreased by CLA feeding, and DHA supplementation did not affect those levels. These results show that DHA supplementation to the CLA diet can attenuate CLA-induced fatty liver through the reduction of hepatic fatty acid synthesis without affecting adipocytokine production in C57BL/6N mice.     

Dietary combination of conjugated linoleic acid (CLA) and pine nut oil prevents CLA-induced fatty liver in mice

Conjugated linoleic acid (CLA) strongly prevents fat accumulation in adipose tissue of mice, even if hepatic fat deposition and insulin resistance are concomitantly observed. This study investigated the possibility of maintaining the antiadiposity properties of CLA while preventing adverse effects such as liver steatosis and hyperinsulinemia. To this end, mice were divided into three groups and fed a standard diet (control) or a diet supplemented with 1% CLA (CLA) or a mixture of 1% CLA plus 7.5% pine nut oil (CLA + P). The combination of CLA + P preserved the CLA-mediated antiadiposity properties (70% fat reduction), preventing hepatic steatosis and a sharp increase in plasmatic insulin starting from the eighth week of CLA treatment. The assay of both fatty acid synthesis and oxidation in the CLA + P mice revealed a time-dependent biphasic behavior of the corresponding enzymatic activities. A sudden change in these metabolic events was indeed found at the eighth week. A strong correlation between the changes in key enzymes of lipid metabolism and in insulin levels apparently exists in CLA-fed mice. Furthermore, lower levels of lipids, in comparison to values found in CLA-fed mice, were observed in the liver and plasma of CLA + P-fed animals.     

Antiatherogenic effects of structured lipid containing conjugated linoleic acid in C57BL/6J mice

Conjugated linoleic acids (CLA) were enzymatically acidolyzed with olive oil to produce structured lipids (SL), and their antiatherosclerotic properties were investigated in C57BL/6J mice. Twenty-eight mice were divided into four groups and fed control diet or atherogenic diets supplemented with high cholesterol and high fat (HCHF) containing 5% of lard, olive oil, or SL based on control diet for 4 weeks. The supplementation of SL diet (0.6% CLA) significantly reduced the levels of serum total cholesterol and total triglyceride and increased high-density lipoprotein cholesterol level as compared to lard and olive oil diet groups (p < 0.05). The activity of liver acyl CoA:cholesterol acyltransferase (ACAT) of mice fed the SL diet was significantly lower than that of mice fed the lard or olive oil diet. A reduced formation of aortic fatty streak was observed in SL group. The extent of CLA incorporation depended on tissues or types of phospholipids. More CLA was incorporated in adipose tissue (1.85 mol %) than in the liver (0.33 mol %). Besides, more CLA was found in phosphatidylethanolamine (PE) (0.47 mol %) than in phosphatidylcholine (PC) (0.05 mol %) of hepatic phospholipids. Hepatic phospholipids (PC and PE) of mice fed the SL diet contained reduced contents of arachidonic and linoleic acid compared with mice fed the olive oil or lard diet. The present study suggests that SL could be considered as a functional oil for preventing risks of atheroscelerosis.     

Influence of feeding soybean oil on conjugated linoleic acid content in beef

Forty-eight steers were used to study the influence of feeding soybean oil (SO) on the conjugated linoleic acid (CLA) content of beef. Steers were fed either a control diet containing 954 g/kg of dry matter (DM) corn-based concentrate (CTL) or a control diet supplemented with SO at 20 (SO2) or 40 g/kg (SO4) of diet DM for 105 days. Adipose tissue samples were collected from the M. longissimus dorsi (LD) and from the M. semitendinosus (ST) on days 0 and 63 of the experiment. Adipose and muscle tissue samples were collected from the LD and ST immediately after slaughter. Feeding 40 g/kg of DM as SO increased the proportions of trans-C(18:1) in beef lipid as compared to CTL and SO2 treatments. The C(18:2) cis-9, trans-11 isomer of CLA as a proportion of total fat was not different in adipose and muscle across treatments. Supplementing SO increased C(18:2) trans-10, cis-12 CLA in adipose tissue of the LD. Supplementing high-grain finishing diets with SO is not an effective strategy to enhance the C(18:2) cis-9, trans-11 isomer of CLA in beef.     

Impact of dietary conjugated linoleic acid on the oxidative stability of rat liver microsomes and skeletal muscle homogenates

Dietary conjugated linoleic acid (CLA; 0-2.0%) increased CLA concentrations in liver microsomes and skeletal muscle homogenates from rats. Dietary CLA decreased oleic and arachadonic acid concentrations in both liver microsomes and skeletal muscle. The presence of CLA in liver microsomes had no impact on linoleic acid, arachadonic acid, and alpha-tocopherol oxidation rates. Dietary CLA (2.0%) also did not alter alpha-tocopherol oxidation rates in liver microsomes or muscle homogenates. Formation of malonaldehyde (MDA) in oxidizing liver microsomes decreased with increasing CLA concentration as determined by measurement of thiobarbituric acid-MDA complexes by HPLC. The ability of CLA to decrease MDA formation without impacting other lipid oxidation markers such as the disappearance of fatty acid and alpha-tocopherol suggests that decreased MDA concentration was the result of CLA's ability to lower polyenoic fatty acids such as arachadonic acid. While CLA does not appear to act as an antioxidant, its ability to decrease polyenoic fatty acid concentrations could decrease the formation of highly cytotoxic lipid oxidation products such as MDA.     

日粮共轭亚油酸对黄羽肉鸡腹脂沉积的影响及机理研究

选择1日龄黄羽肉鸡60只,随机分成2组。在实验组中添加3%共轭亚油酸(conjugated linoleic acid,CLA),对照组日粮中添加3%的食用菜籽油。49日龄屠宰,采集腹部脂肪组织。用RT-PCR方法,以β-actin为内标,相对定量测定腹脂中鸡生长激素受体（cGHR）、胰岛素样生长因子-1（cIGF-1）、胰岛素样生长因子Ⅰ型受体（cIGF-ⅠR）、过氧化物增殖剂活化受体γ（cPPARγ）、脂联素（cAdiponectin）及其Ⅰ型受体（cAdipoⅠR）的mRNA丰度。结果显示：CLA处理使肉鸡的腹脂沉积下降了20.93%（P<0.05）,分别使腹脂中鸡cGHR mRNA和cPPARγ mRNA降低了24.74%（P<0.05）和66.52%（P<0.01）;而对cIGF-1、cIGF-ⅠR、cAdiponectin、cAdipoⅠR 基因表达无显著影响（P>0.05）;数据提示：CLA降低肉鸡脂肪沉积的作用可能通过GH途径和PPARγ通路介导,而与IGF-1和（或）GH-IGF-1途径、Adiponectin 的作用无关。     

Effect of dietary conjugated linoleic acid on lipid peroxidation and histological change in rat liver tissues

The effect of dietary conjugated linoleic acid (CLA) on hepatic lipid parameters in Sprague-Dawley rats was examined. When rats were fed a diet containing CLA at 0 (control), 1, or 2% of the weight of the amount of food given for 3 weeks, the liver weight exhibited a slight increase in the CLA-fed groups, although the difference was not significant. Lipid accumulation in the hepatocytes of CLA-fed rats was also demonstrated by electron microscopic observation. In addition, the liver thiobarbituric acid reactive substances levels were significantly higher in the 2 wt % CLA group than in the other two dietary groups, and the levels of phosphatidylcholine hydroperoxide were higher in CLA-fed groups when compared to that of the control group. On the other hand, the serum lipid peroxide levels were comparable among all three dietary groups. Levels of triglycerides in the white adipose tissue (WAT) and serum nonesterified fatty acid (NEFA) were reduced in a CLA-dose-dependent manner. CLA was shown to accumulate in the WAT much more than in the serum or liver. These results suggest that CLA accelerates the decomposition of storage lipids in WAT and the clearance of serum NEFA levels, resulting in lipid peroxidation and a morphological change in the liver.     

Enrichment of conjugated linoleic acid in oats (Avena sativa L.) by microbial isomerization

A method for microbial isomerization of oat linoleic acid to conjugated linoleic acid (CLA) was developed. The method includes hydrolysis of oat lipids in aqueous flour slurries by the endogenous oat lipase. Then, the flour slurry containing free linoleic acid is utilized as a substrate for the isomerization reaction carried out by resting cells of Propionibacterium freudenreichii ssp. shermanii. The isomerization reaction progressed most effectively when, after the lipid hydrolysis period, the pH of the slightly acidic oat slurry was elevated to 8.0-8.5 and maintained at this range. With slurries containing 5% (w/v) oat flour, the amounts of CLA formed per dry matter were up to 10.1 mg/g corresponding to 102 mg/g lipids or 0.44 mg/mL slurry. Increments in the flour content up to 15% increased the volumetric production of CLA to 0.85 mg/mL. The proportion of the cis-9,trans-11 isomer was 80% of the total CLA formed. CLA could be concentrated into the solid material of the oat slurry by acidification.     

Comparison of different methylation methods for the analysis of conjugated linoleic acid isomers by silver ion HPLC in beef lipids

Four different methods for the methylation of conjugated linoleic acid isomers (CLA) in ruminant lipids were compared by silver ion (Ag+) HPLC. The combination of base-catalyzed methods followed by an acid-catalyzed method with BF3/MeOH was tested under different temperatures (room temperature and at 60 degrees C), along with based-catalyzed methylation with NaOCH3 and methylation with BF3/MeOH after saponification with NaOH. The comparison among these four methods was done on muscle and adipose tissue samples from bulls. The repeatability theta of the combined base- and acid-catalyzed methylation (NaOCH3/BF3) at ambient temperature for 20 min and at 60 degrees C for 10 min was most suitable for the quantitative Ag+-HPLC analysis of CLA isomers. At 60 degrees C the combined methods supplied the highest concentrations of most CLA isomers. The base-catalyzed methylation and the saponification followed by BF3/MeOH methylation for 5 min generated significantly lower concentrations for most CLA isomers compared to the combined methods.     

Effects of oleic acid rich oils on aorta lipids and lipoprotein lipase activity of spontaneously hypertensive rats

Hypertension development in the spontaneously hypertensive rat (SHR) leads to vascular wall widening by smooth muscle cell proliferation. In these cells, triglycerides (TG) and cholesteryl esters (CE) can accumulate until they become foam cells. We administrated two oleic rich oils, virgin olive (VOO) and high oleic sunflower oils (HOSO), to Wistar-Kyoto rats (WKY) and SHR because these oils have been reported to reduce the risk for coronary heart disease in hypertensive patients and SHR. After 12 weeks of feeding, we analyzed the TG and CE composition and the lipolytic (lipoprotein lipase, LPL, and non-LPL) activity in aortas of these animals. HOSO increased the content of linoleic acid in CE and TG of aortas from both WKY and SHR as compared with animals fed VOO by proportionally decreasing the content of oleic acid. Conversely, VOO reduced the LPL and non-LPL lipolytic activities, hence limiting the free fatty acids available for the synthesis of TG and CE in the vascular wall.     

Acidolysis of tristearin with selected long-chain fatty acids

Five lipases, namely, Candida antarctica (Novozyme-435), Mucor miehei (Lipozyme-IM), Pseudomonas sp. (PS-30), Aspergillus niger (AP-12), and Candida rugosa (AY-30), were screened for their effect on catalyzing the acidolysis of tristearin with selected long-chain fatty acids. Among the lipases tested C. antarctica lipase catalyzed the highest incorporation of oleic acid (OA, 58.2%), gamma-linolenic acid (GLA, 55.9%), eicosapentaenoic acid (EPA, 81.6%), and docosahexaenoic acid (DHA, 47.7%) into tristearin. In comparison with other lipases examined, C. rugosa lipase catalyzed the highest incorporation of linoleic acid (LA, 75.8%), alpha-linolenic acid (ALA, 74.8%), and conjugated linoleic acid (CLA, 53.5%) into tristearin. Thus, these two lipases might be considered promising biocatalysts for acidolysis of tristearin with selected long-chain fatty acids. EPA was better incorporated into tristearin than DHA using the fifth enzymes. LA incorporation was better than CLA. ALA was more reactive than GLA during acidolysis, except for the reaction catalyzed by Pseudomonas sp., possibly due to structural differences (location and geometry of double bonds) between the two fatty acids. In another set of experiments, a combination of equimolar quantities of unsaturated C18 fatty acids (OA + LA + CLA + GLA + ALA) was used for acidolysis of tristearin to C18 fatty acids at ratios of 1:1, 1:2, and 1:3. All lipases tested catalyzed incorporation of OA and LA into tristearin except for M. miehei, which incorportaed only OA. C. rugosa lipase better catalyzed incorporation of OA and LA into tristearin than other lipases tested, whereas the lowest incorporation was obtained using Pseudomonas sp. As the mole ratio of substrates increased from 1 to 3, incorporation of OA and LA increased except for the reaction catalyzed by A. niger and C. rugosa. All lipases tested failed to allow GLA or CLA to participate in the acidolysis reaction, and ALA was only slightly incoporated into tristearin when M. miehei was used.     

Isomeric distribution of conjugated linoleic acids (CLA) in the tissues of layer hens fed a CLA diet

The isomeric distribution of conjugated linoleic acids (CLA) in the tissue lipids of hens in relation to that in the diet was examined. Silver-ion high-performance liquid chromatography was used to quantify individual CLA isomers in total tissue lipids, phospholipids, and triacylglycerols. It was found that the deposition of CLA isomers in hen tissues was selective. All tissues including serum, liver, heart, kidney, abdominal fat, and leg and breast muscles had lesser amounts of total cis/trans isomers ranging from 75.87 to 89.13% of total CLA, which was in contrast to the value of 92% of total CLA in the dietary lipids. Total trans/trans isomers in all tissue lipids ranging from 6.11 to 18.02% of total CLA were greater than that in the diet (4.19%). Among the individual trans/trans isomers, all tissues except for adipose tissue and brain incorporated greater amounts of t-12,t-14-18:2, t-11,t-13-18:2,t-10,t-12-18:2, t-9,t-11-18:2, and t-18,t-10-18:2 compared with the values of the diet. Within the cis/trans group, lesser amounts of c-10,t-12/t-10,c-12-18:2 were found to incorporate into all tissues compared with the value of the diet. Serum and liver had higher percentages of c-9,t-11/t-9,c-11, whereas the other tissues had similar levels of this isomer compared with that of the diet. It was also observed that supplementation of CLA in the diet of layer hens decreased the concentration of docosahexaenoic acid (22:6n-3) in all of the tissue lipids. It is concluded that dietary CLA can transfer to the tissue but that incorporation of CLA isomers into the tissue is selective in hens.     

Effect of antioxidants on soy oil conjugated linoleic acid production and its oxidative stability

Conjugated linoleic acid (CLA)-rich soy oil can be produced by photoisomerization of soy oil linoleic acid to produce a soy oil with up to 20% CLA. Recent studies indicate that mixed soy tocopherols added to refined bleached deodorized (RBD) oil produced significant increase in soy CLA yield during soy oil linoleic acid photoisomerization. However, the effect of common synthetic free radical scavenging antioxidants and specific tocopherols on CLA yield and its oxidative stability is not known. Therefore, this investigation evaluated the effects of various antioxidant systems on soy oil CLA yield and oxidative stability. Soy oil with added antioxidants consisting of combinations of mixed tocopherols (MT), ascorbyl palmitate (AP), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ) was photoisomerized to produce CLA- rich soy oil. The CLA content was determined by GC-FID analysis and oxidative stability by peroxide value (PV). The soy oil in the presence of TBHQ, MT alone and MT with 500 ppm of AP produced significantly greater CLA yields and improved oxidative stability compared to a control without added antioxidants (p < 0.05). However, added mixed tocopherols produced the greatest CLA yield and also reduced PV relative to the control. Tocopherols in the form of α-, γ- and δ-tocopherols were then each examined as to their relative effect on CLA yields and PV. The largest increase in CLA yield was obtained with 1800 ppm of γ-tocopherols with reduced PV. Mixed tocopherols, TBHQ and γ-tocopherols can be used to increase CLA yield and reduce PV of soy oil during linoleic acid photoisomerization.     

Incorporation of alpha-tocopherol and linoleic acid in fresh lambs by feeding chemically treated dietary supplements containing dl-alpha-tocopheryl acetate and sunflower oil

The effects of feeding chemically treated dietary supplements (CTDS) containing sunflower oil and dl-alpha-tocopheryl acetate (TA) on alpha-tocopherol content and fatty acid profile in edible tissues of lambs were estimated. Compared with lambs fed control diet (CD), lambs fed CD plus 250 IU of either TA or CTDS increased serum alpha-tocopherol. The CTDS-fed lambs further increased serum alpha-tocopherol by 29% over those fed CD plus 250 IU of TA. Lambs supplemented with TA or CTDS increased alpha-tocopherol in muscle and adipose tissues as compared with lambs fed CD. The CTDS-fed lambs had higher levels of alpha-tocopherol in gluteus medius (7.55 vs 6.05 mug/g), psoas major (7.43 vs 6.02 mug/g), and subcutaneous fat (12.6 vs 9.98 mug/g) compared with the TA-fed lambs. Feeding lambs CTDS also substantially increased levels of linoleic acid in the adipose tissues while decreasing the content of palmitic and oleic acids.     

Effect of conjugated linoleic acids on the activity and mRNA expression of 5- and 15-lipoxygenases in human macrophages

Lipoxygenases are a family of non-heme enzyme dioxygenases. The role of lipoxygenases is synthesis of hydroperoxides of fatty acids, which perform signaling functions in the body. Studies on conjugated linoleic acids (CLAs) as fatty acids with a potential anti-atherosclerotic function have recently been initiated. The aim of the study was to test the effect of CLAs and linoleic acid on 5- and 15-lipoxygenase (5-LO, 15-LO-1) enzyme activity, their mRNA expression, and concentration in the cells. It was also desired to determine whether the CLAs are substrates for the enzymes. For the experiments monocytic cell line (THP-1) and monocytes obtained from human venous blood were used. Monocytes were differentiated to macrophages: THP-1 (CD14+) by PMA administration (100 nM for 24 h) and monocytes from blood (CD14+) by 7-day cultivation with the autologous serum (10%). After differentiation, macrophages were cultured with 30 microM CLAs or linoleic acid for 48 h. The 15- and 5-lipoxygenase products were measured by HPLC method. mRNA expression and protein content were analyzed by real-time PCR and Western blot analysis. The in vitro studies proved that both CLA isomers are not substrates for 15-LO-1; in ex vivo studies hydroxydecadienoic acid (HODE) concentration was significantly reduced (p = 0.019). The trans-10,cis-12 CLA isomer reduced HODE concentration by 28% (p = 0.046) and the cis-9,trans-11 CLA isomer by 35% (p = 0.028). In macrophages obtained from THP-1 fatty acids did not change significantly mRNA expression of the majority of the investigated genes. CLAs did not change the content of 5-LO and 15-LO-1 proteins in macrophages obtained from peripheral blood. Linoleic acid induced 15-LO-1 expression (2.6 times, p < 0.05). CLAs may perform the function of an inhibitor of lipoxygenase 15-LO-1 activity in macrophages.     

C-terminal region of Candida rugosa lipases affects enzyme activity and interfacial activation

Candida rugosa contains several lipase (CRLs) genes, and CRLs show diverse enzyme activity despite being highly homologous across their entire protein family. Previous studies found that LIP4 has a high esterase activity and a low lipolytic activity and lacks interfacial activation. To investigate whether the C-terminal region of the CRLs mediates enzymatic activity, chimeras were generated in which the C-terminus of LIP4 from either residue 374, 396, 417, or 444 to residue 534 was swapped with the corresponding peptide from the isoform LIP1. A chimeric lipase containing the C-terminus from 396 to 534 of LIP1 on a LIP4 scaffold showed activity similar to that of commercial CRL on triolein, and lipolytic activity increased 2-6-fold over that of LIP4. Moreover, interfacial activation was also observed in the chimeric lipase. To improve its enzymatic properties, a novel glycosylation site was added at residue 314. The new glycosylated lipase showed improved thermostability and enhancement in enzymatic activity, indicating its potential for use in further application.     

Functional proteomic analysis of rice bran esterases/lipases and characterization of a novel recombinant esterase

An esterase from rice ( Oryza sativa ) bran was identified on two-dimensional gel using 4-methylumbelliferyl butyrate as a substrate. The esterase cDNA (870 bp) encoded a 289 amino acid protein (designated OsEST-b) and was expressed in Escherichia coli . The molecular weight of recombinant OsEST-b (rOsEST-b) was 27 kDa, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemical characterization demonstrated that rOsEST-b was active over a broad temperature range (optimum at 60 °C) and preferred alkaline conditions (optimum at pH 9.0). The rOsEST-b showed maximum activity toward p-nitrophenyl butyrate (C(4)) among various p-nitrophenyl esters (C(4)-C(18)), indicating that rOsEST-b is an esterase for short-chain fatty acids. The kinetic parameters under optimal conditions were K(m) = 27.03 μM, k(cat) = 49 s(-1), and k(cat)/K(m) = 1.81 s(-1) μM(-1). The activity of rOsEST-b was not influenced by ethylenediaminetetraacetic acid, suggesting that it is not a metalloenzyme. The amino acid sequence analysis revealed that OsEST-b had a conserved pentapeptide esterase/lipase motif but that the essential active site serine (GXSXG) was replaced by cysteine (C). These results suggest that OsEST-b is distinct from traditional esterases/lipases and is a novel lipolytic enzyme in rice bran.