Hepatic lipid characteristics and histopathology of laying hens fed CLA or n−3 fatty acids

The effect of dietary CLA and n−3 PUFA on hepatic TAG accumulation, histopathology, and FA incorporation in lipid classes by laying chickens was investigated. One hundred twenty 30-wk-old single-comb white leghorn laying hens were distributed randomly to four treatments (3 replications of 10 birds) and were fed diets containing CLA and animal fat (Diet I), 18∶3n−3 (Diet II), or long-chain n−3 FA (Diet III). A sunflower oil (n−6 FA)-based diet was the control. Feeding Diet I resulted in an increase in hepatic total lipids (P<0.05). The liver TAG content was 32.2, 18.9, 29.4, and 18.7 mg/g for hens fed Diet I, Diet II, Diet III, and the control diet, respectively (P<0.05). The serum TAG was lowest in bilds fed Diet II (P<0.05). Diet I resulted in an increase in the total number of fat vacuoles and lipid infiltration in hepatocytes (P<0.05). The number of cells with 75% or higher lipid vacuolation was observed only in birds fed Diet I. Feeding diets containing CLA resulted in an increase in the content of the c9,t11 CLA isomer in liver TAG and PC (P<0.05). No difference was observed in the CLA concentration of hepatic PE fractions. The content of DHA (22∶6n−3) was higher in the TAG, PC, and PE of hens fed Diet II and Diet III than Diet I and the control (P<0.05). Feeding CLA resulted in an increase in total saturated FA in the TAG and PC fractions (P<0.05). Long-term feeding of CLA in laying birds leads to an increase in liver TAG and may predispose birds to fatty liver hemorrhagic syndrome.     

Production of Human Milk Fat Substitutes by Interesterification of Tripalmitin with Ethyl Oleate Catalyzed by Candida parapsilosis Lipase/Acyltransferase

In human milk fat, the saturated fatty acids, namely palmitic acid, are located at the sn-2 position of triacylglycerols (TAG) while unsaturated fatty acids (e.g. oleic acid) are esterified at position sn-1,3. Thus, sn-1,3-dioleoyl-2-palmitoylglycerol (OPO) is the target TAG to be used as human milk fat substitutes (HMFS) in infant formulas. In this study, the noncommercial recombinant lipase/acyltransferase from Candida parapsilosis (CpLIP2) was immobilized in Accurel MP1000, and used as a biocatalyst for the interesterification of tripalmitin with ethyl oleate in a solvent-free medium, to obtain structured lipids used as HMFS. Different molar ratios (MR) of ethyl oleate to tripalmitin (2:1–8:1) were used. After 4 h reaction at 60°C, about 30 mol% of oleic acid incorporation was already observed for all tested MR. An apparent equilibrium was reached after 8–24 h, with 32–51 mol% final incorporation, increasing with the MR. The incorporation of oleic acid into TAG was compared with the maximum predicted values when a random or a sn-1,3-regioselective biocatalyst was used. The obtained values are consistent with the maximum incorporation expected for a sn-1,3-regioselective enzyme. In fact, the amount of oleic acid at position sn-2 was approximately 15% for all the MR tested, which is explained by the acyl migration phenomenon. CpLIP2 exhibited higher activity than most commercial immobilized lipases (e.g. faster reaction in solvent-free media, low enzyme load, and low MR needed), and showed a recognized sn-1,3 regioselective behavior.     

Synthesis and Characterization of Soyamide Ferulate

Ferulic acid (4-hydroxy-3-methoxycinnamic acid) is a phytochemical antioxidant that is widely distributed throughout the plant kingdom. Cinnamic acid derivatives are used as biobased ultraviolet (UV) absorbers in sunscreen formulations. Soybean oil ferulate, a biobased UV absorber, was synthesized by reacting soyamide with ferulic acid. The resulting product was characterized by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopy, and gel permeation chromatography (GPC). Spectroscopic studies measured the soyamide-based ferulate maximum absorbance at 327 nm with a molar extinction coefficient of 19,705 L mol⁻¹ cm⁻¹. Soyamide ferulate has potential to be used in coatings.     

Effect of CLA on milk fat synthesis in dairy cows: Comparison of inhibition by methyl esters and free fatty acids,and relationships among studies

CLA is a potent inhibitor of milk fat synthesis, as shown by investigations using mixtures of CLA isomers in FFA form. However, methyl esters of CLA can be initially formed in commercial synthesis, and their use in a supplement has certain manufacturing and cost advantages. Our objective was to compare abomasal infusion of methyl esters of CLA (ME-CLA) and FFA of CLA (FFA-CLA) on milk fat synthesis. Data were also combined with previous investigations to examine broader relationships between trans-10,cis-12 CLA and the reduction in milk fat. Three mid-lactation, rumen-fistulated Holstein cows were used in a 3×3 Latin square design. Treatments were (i) control, (ii) ME-CLA, and (iii) FFA-CLA. The ME-CLA and FFA-CLA treatments (4.2 g/d trans-10,cis-12 CLA) resulted in a comparable reduction in milk fat yield (38 and 39%, respectively) and pattern of reduction in individual FA. In contrast, milk yield, milk protein, and feed intake were unaltered by CLA treatment. Combining data across studies revealed strong correlations relating the reduction in milk fat yield to abomasal dose of trans-10,cis-12 CLA (R ²=0.86), milk fat content of trans-10,cis-12 CLA (R ²=0.93), and milk fat secretion of trans-10,cis-12 CLA (R ²=0.82). Across studies, transfer efficiency of abomasally infused trans-10,cis-12 CLA into milk fat was relatively constant (22%; R ²=0.94). Overall, ME-CLA and FFA-CLA were equally potent in reducing milk fat, and either form could be used to formulate a dietary supplement that would induce milk fat depression.     

Fatty acid and conjugated linoleic acid isomer profiles in human milk fat

The fatty acid composition of 39 mature human milk samples from four Spanish women collected between 2 and 18 weeks during lactation was studied by gas chromatography. The conjugated linoleic acid (CLA) isomer profile was also determined by silver‐ion HPLC (Ag⁺‐HPLC) with three columns in series. The major fatty acid fraction in milk lipids throughout lactation was represented by the monounsaturated fatty acids, with oleic acid being the predominant compound (36–49% of total fatty acids). The saturated fatty acid fraction represented more than 35% of the total fatty acids, and polyunsaturated fatty acids ranged on average between 10 and 13%. Mean values of total CLA varied from 0.12 to 0.15% of total fatty acids. The complex mixture of CLA isomers was separated by Ag⁺‐HPLC. Rumenic acid (RA, cis‐9 trans‐11 C18:2) was the major isomer, representing more than 60% of total CLA. Trans‐9 trans‐11 and 7‐9 (cis‐trans + trans‐cis) C18:2 were the main CLA isomers after RA. Very small amounts of 8‐10 and 10‐12 C18:2 (cis‐trans + trans‐cis) isomers were detected, as were different proportions of cis‐11 trans‐13 and trans‐11 cis‐13 C18:2. Although most of the isomers were present in all samples, their concentrations varied considerably.     

Positional distribution of CLA in TAG of lamb tissues

The content and positional distribution of CLA in TAG fractions of lamb tissues was examined with either preformed CLA or the linoleic acid precursor of CLA in the diet as experimental treatments. The CLA content of phospholipid (PL) from these tissues was also examined. Thirteen lambs were randomized to the following dietary treatments: (i) control diet (no supplement); (ii) CLA supplementation (0.33 g d⁻¹ for 21 d prior to weaning) to milk-replacer of pre-ruminating lambs, or (iii) feeding linoleic acid-rich oil (6% safflower oil on a dry matter basis) to weaned ruminating lambs. At slaughter, tissue samples were procured from diaphragm, rib muscle, and subcutaneous (SC) adipose tissue. Safflower oil supplementation in the diet resulted in an increase in CLA content of the TAG from diaphragm, rib muscle, and SC adipose tissue by about threefold (P<0.05) on a mol% basis. CLA was localized to the sn-1/3 positions of TAG. Animals that received pre-formed CLA, however, had increased proportions of CLA at the sn-2 position of TAG from SC adipose tissue, suggesting that there were tissue-specific dietary effects and possible age-related effects on the mode of FA incorporation into TAG. Safflower oil supplementation in the diet had no effect on the CLA content of PL from diaphragm, rib muscle, and SC adipose tissue, suggesting that CLA was preferentially incorporated into the TAG of these tissues.     

Maternal dietary conjugated linoleic acid alters hepatic triacylglycerol and tissue fatty acids in hatched chicks

The effects of feeding CLA to hens on newly hatched chick hepatic and carcass lipid content, liver TAG accumulation, and FA incorporation in chick tissues such as liver, heart, brain, and adipose were studied. These tissues were selected owing to their respective roles in lipid assimilation (liver), as a major oxidation site (heart), as a site enriched with long-chain polyunsaturates for function (brain), and as a storage depot (adipose). Eggs with no, low, or high levels of CLA were produced by feeding hens a corn-soybean meal-basal diet containing 3% (w/w) corn oil (Control), 2.5% corn oil +0.5% CLA oil (CLA1), or 2% corn oil +1.0% CLA oil (CLA2). The egg yolk content of total CLA was 0.0, 1.0, and 2.6% for Control, CLA1, and CLA2, respectively (P<0.05). Maternal dietary CLA resulted in a decrease in chick carcass total fat (P<0.05). Liver tissue of CLA2 chicks had the lowest fat content (P<0.05). The liver TAG content was 8.2, 5.8, and 5.1 mg/g for Control, CLA1, and CLA2 chicks, respectively (P<0.05). The chicks hatched from CLA1 and CLA2 incorporated higher levels of cis-9,trans-11 CLA in the liver, plasma, adipose, and brain than Control (P<0.05). The content of 18∶0 was higher in the liver, plasma adipose, and brain of CLA1 and CLA2 than Control (P<0.05), but no difference was observed in the 18∶0 content of heart tissue. A significant reduction in 18∶1 was observed in the liver, plasma, adipose, heart, and brain of CLA1 and CLA2 chicks (P<0.05). DHA (22∶6n−3) was reduced in the heart and brain of CLA1 and CLA2 chicks (P<0.05). No difference was observed in carcass weight, dry matter, or ash content of chicks (P>0.05). The hatchabilities of fertile eggs were 78, 34, and 38% for Control, CLA1, and CLA2, respectively (P<0.05). The early dead chicks were higher in CLA1 and CLA2 than Control (18 and 32% compared with 9% for Control), and alive but not hatched chicks were 15 and 19% for CLA1 and CLA2, compared with 8% for Control (P<0.05). Maternal supplementation with CLA leads to a reduction in hatchability, liver TAG, and carcass total fat in newly hatched chicks.     

Changes in the milk and cheese fat composition of ewes fed commercial supplements containing linseed with special reference to the CLA content and isomer composition

A study was carried out to increase the CLA contents in ewes’ milk fat under field conditions by dietary means and to investigate the extent of the changes and consequences for milk processing and cheese quality. During a 3-mon period, ewes’ bulk milk samples were collected every week from two different herds. For the first 4 wk the ewes were fed a conventional diet. Then the following 6 wk a supplement enriched in α-linolenate (whole linseed) was incorporated into the ovine diet. Finally, in the last 3 wk the feeding was the same as in the first 4 wk. The FA profile in milk fat was monitored by GC, and the distribution of CLA isomers was thoroughly tested by combining GC-MS of 4,4-dimethyloxazoline derivatives (DMOX) with silver ion-HPLC (Ag⁺-HPLC) of FAME. Reconstructed mass spectral profiles of CLA characteristic ions from DMOX were used to identify positional isomers, and Ag⁺-HPLC was used to quantify them. An increase in total CLA in milk fat was observed, and total CLA remained elevated during the weeks of enriched α-linolenate feeding. In our experimental conditions there was a linear relationship between trans-vaccenic acid (trans-11-octadecenoic acid; trans-11 18∶1) and 9-cis, 11-trans CLA in ewes’ milk fat. Concerning the CLA isomer profile, increases in the 11,13- and 12,14–18∶2 positional isomers were considerable when linseed was included in the diet. Organoleptic characteristics of cheeses made with CLA-enriched milk did not substantially differ from those made with nonsupplemented ewes’ milk. CLA total content and isomer profile did not change during ripening.     

Effect of abomasal infusions of geometric isomers of 10,12 conjugated linoleic acid on milk fat synthesis in dairy cows

The trans-10, cis-12 isomer of conjugated linoleic acid (CLA) decreases TAG accumulation in 3T3-L1 adipocytes, reduces lipid accretion in growing animals, and inhibits milk fat synthesis in lactating mammals. However, there is evidence to suggest that other FA may also exert antilipogenic effects. In the current experiment, the effects of geometric isomers of 10,12 CLA on milk fat synthesis were examined using four Holstein-British Friesian cows in a 4×4 Latin Square experiment with 14-d periods. Treatments consisted of abomasal infusions of skim milk, or skim milk containing trans-10, cis-12 CLA (T1), trans-10, trans-12 CLA (T2), or a mixture of predominantly 10,12 isomers containing (g/100 g) trans-10, cis-12 (35.0), cis-10, trans-12 (23.2), trans-10, trans-12 (14.9), and cis-10, cis-12 (5.1). CLA supplements were prepared from purified ethyl linoleate and infused as nonesterified FA. Infusions were conducted over a 4-d period with a 10-d interval between treatments and targeted to deliver 4.5 g/d of 10,12 CLA isomers. Compared with the control, trans-10, trans-12 CLA had no effect (P>0.05) on milk fat yield, whereas treatments T1 and T3 depressed (P<0.05) milk fat content (19.8 and 22.9%, respectively) and decreased milk fat output (20.8 and 21.3%, respectively). Comparable reductions in milk fat synthesis to 4.14 and 1.80 g trans-10, cis-12/d supplied by treatments T1 and T3 indicate that other 10,12 geometric isomers of CLA have the potential to exert antilipogenic effects. The relative abundance of cis-10, trans-12 CLA in treatment T3 and the low transfer efficiency of this isomer into milk suggest that cis-10, trans-12 CLA was the active component.     

Modifying milk fat composition of dairy cows to enhance fatty acids beneficial to human health

There is increased consumer awareness that foods contain microcomponents that may have beneficial effects on health maintenance and disease prevention. In milk fat these functional food components include EPA, DHA, and CLA. The opportunity to enhance the content of these FA in milk has improved as a result of recent advances that have better defined the interrelationships between rumen fermentation, lipid metabolism, and milk fat synthesis. Dietary lipids undergo extensive hydrolysis and biohydrogenation in the rumen. Milk fat is predominantly TG, and de novo FA synthesis and the uptake of circulating FA contribute nearly equal amounts (molar basis) to the FA in milk fat. Transfer of dietary EPA and DHA to milk fat is very low (<4%); this is, to a large extent, related to their extensive biohydrogenation in the rumen, and also partly due to the fact that they are not transported in the plasma lipid fractions that serve as major mammary sources of FA uptake (TG and nonesterified FA). Milk contains over 20 isomers of CLA but the predominant one is cis-9,trans-11 (75–90% of total CLA). Biomedical studies with animal models have shown that this isomer has anticarcinogenic and anti-atherogenic activities. cis-9,trans-11-CLA is produced as an intermediate in the rumen biohydrogenation of linoleic acid but not of linolenic acid. However, it is only a transient intermediate, and the major source of milk fat CLA is from endogenous synthesis. Vaccenic acid, produced as a rumen biohydrogenation intermediate from both linoleic acid and linolenic acid, is the substrate, and Δ9-desaturase in the mammary gland and other tissues catalyzes the reaction. Diet can markedly affect milk fat CLA content, and there are also substantial differences among individual cows. Thus, strategies to enhance milk fat CLA involve increasing rumen outflow of vaccenic acid and increasing Δ9-desaturase activity, and through these, several-fold increases in the content of CLA in milk fat can be routinely achieved. Overall, concentrations of CLA, and to a lesser extent EPA and DHA, can be significantly enhanced through the use of diet formulation and nutritional management of dairy cows.     

Supplementation with CLA: Isomer incorporation into serum lipids and effect on body fat of women

Animal studies have suggested that CLA, a natural component of meat and dairy products, may confer beneficial effects on health. However, human studies using supplementation with CLA have produced contradictory results. The aim of the present study was to further investigate the effect of CLA supplementation on human body fat, serum leptin, and serum lipids, as well as the incorporation of CLA isomers into serum lipids classes. Sixteen young healthy nonobese sedentary women received 2.1 g of CLA (divided equally between the cis,trans-9,11 and trans,cis-10,12 isomers) daily for 45 d and placebo for 45 d in a randomized double-blind crossover design. Body fat was estimated (by measurement of skinfold thickness at 10 sites), and blood was sampled at the beginning, middle, and end of the entire intervention period; an additional blood sample was obtained 2 wk thereafer. No significant differences in energy, carbohydrate, lipid, or protein intake existed between the CLA and placebo intake periods. No significant differences were found in body fat or serum leptin, TAG, total cholesterol, HDL-cholesterol, and alanine aminotransferase between CLA and placebo. The CLA isomer content of serum TAG, phospholipids, and total lipids increased 2–5 times with CLA supplementation (P<0.05). In contrast, the CLA content of cholesteryl esters did not change significantly. The period of 2 wk after the end of CLA supplementation was sufficient for its washout from serum lipids. These data indicate that supplementation with 2.1 g of CLA daily for 45 d increased its levels in blood but had no effect on body composition or the lipidemic profile of nonobese women.     

Two-Step Production of Oil Enriched in Conjugated Linoleic Acids and Diacylglycerol

A two-step process was used to produce diacylglycerol-enriched structured lipid that contained mainly c9,t11 and t10,c12 isomers of conjugated linoleic acids (CLA). First, a structured triacylglycerol (TAG) was synthesized by lipase-catalyzed acidolysis of corn oil with CLA. This structured triacylglycerol contained 30.4 mol% CLA with 45.5% of the CLA mostly located at sn-1,3 positions of the glycerol backbone. Then, lipase-catalyzed glycerolysis was conducted between structured triacylglycerol and glycerol to produce diacylglycerol-enriched structured lipid. The final product contained 6.8% monoacylglycerol, 31.5% diacylglycerol and 61.1% TAG after 48 h reaction. The selected chemical (fatty acid composition, the content of mono-, di-, and triacylglycerol in the reaction product) and physical properties (melting profile) were determined by hihg-performance liquid chromatography (HPLC), gas chromatography (GC), and differential scanning calorimetry (DSC).     

Synthesis of the structured lipid 1,3-dioleoyl-2-palmitoylglycerol from palm oil

Human milk fat contains 20–25% palmitic acid (16∶0) and 30–35% oleic acid (18∶1). More than 60% of the plamitic acid occurs at the sn-2 position of the glycerol backbone. Palm oil is a rich source of both palmitic and oleic acids. The structured lipid 1,3-dioleyl-2-palmitoylglycerol (OPO) is an important ingredient in infant formula. OPO was synthesized from palm oil by a three-step method. In the first step, low-temperature fractionation was applied to palm oil FA, yielding a palmitic acid-rich fraction (87.8%) and an oleic acid-rich fraction (96%). The palmitic acid content was further increased to 98.3% by transforming palmitic acid into ethyl palmitate. In the second step, esterification of ethyl palmitate and glycerol catalyzed by lipase Novozym 435 under vacuum (40 mm Hg) was employed for the synthesis of tripalmitin. Finally, OPO was obtained by the reaction of tripalmitin. Finally, OPO was obtained by the reaction of tripalmitin with oleic acid catalyzed by Lipase IM 60. In this final step, the TAG content in the product acylglycerol mixture was 97%, and 66.1% oleic acid was incorporated into TAG. Analysis of the FA composition at the sn-2 position of TAG showed 90.7 mol% of palmitic acid and 9.3 mol% of oleic acid. OPO content in the product TAG was ca. 74 mol%. Thus, an efficient method was developed for the synthesis of OPO from palm oil.     

Quantitative determination of conjugated linoleic acid isomers in beef fat

The amounts of 14 conjugated linoleic acid (CLA) isomers (t12t14, t11t13, t10t12, t9t11, t8t10, t7t9, t6t8; 12,14 c/t, t11c13, c11t13, t10c12, 9,11 c/t, t8c10, t7c9‐18:2) in 20 beef samples were determined by triple‐column silver‐ion high‐performance liquid chromatography (Ag⁺‐HPLC). Quantitation was performed using an external CLA reference standard consisting of cis9,trans11‐18:2,trans9,trans11‐18:2 and cis9,cis11‐18: 2. Linearity was checked as being r > 0.9999 between 0.02 × 10^‐3 to 2 mg/ml. The determination limit (5‐fold signal/noise ratio) of the CLA reference was estimated to be 0.25, 0.50, 1.0 ng/injection for the cis/trans, trans,trans and cis,cis isomers, respectively. As expected, cis9,trans11‐18:2 was the predominant isomer (1.95 ± 0.54 mg/g fat) in beef, followed by trans7,cis9‐18:2 (0.19 ± 0.04 mg/g fat); cis,cis isomers were below the determination limit in most beef samples. Total CLA amounts determined by Ag⁺‐HPLC were compared to total CLAs determined by gas chromatography (GC, 100 m CPSil^TM 88 column). The amounts obtained by GC were generally higher than those determined by Ag⁺ ‐HPLC due to co‐eluting compounds.     

Triacylglycerol composition and structure in genetically modified sunflower and soybean oils

Changes in composition were examined in oils extracted from genetically modified sunflower and soybean seeds. Improvements were made to the analytical methods to accomplish these analyses successfully. Triacylglycerols (TAG) were separated on two 300 mm × 3.9 mm 4μ Novapak C18 high-performance liquid chromatography (HPLC) columns and detected with a Varex MKIII evaporative light-scattering detector. Peaks were identified by coelution with known standards or by determining fatty acid composition of eluted TAG by capillary gas chromatography (GC). Stereospecific analysis (fatty acid position) was accomplished by partially hydrolyzing TAG with ethyl magnesium bromide and immediately derivatizing the resulting diacylglycerols (DAG) with (S)-(+)-1-(1-naphthyl)ethyl isocyanate. The derivatized sn-1,2-DAG were completely resolved from the sn-2,3-DAG on two 25 mm × 4.6 mm 3 μ silica HPLC columns. The columns were chilled to −20°C to obtain baseline resolution of collected peaks. The distribution of fatty acids on each position of the glycerol backbone was derived from the fatty acid compositions of the two DAG groups and the unhydrolyzed oil. Results for the sn-2 position were verified by hydrolyzing oils with porcine pancreatic lipase, isolating the resulting sn-2 monoacylglycerols by TLC, and determining the fatty acid compositions by GC. Results demonstrated that alterations in the total fatty acid composition of these seed oils are determined by the concentration of TAG species that contain at least one of the modified acyl groups. As expected, no differences were found in TAG with fatty acid quantities unaffected by the specific mutation. In lieu of direct metabolic or enzymatic assay evidence, the authors’ positional data are nevertheless consistent with TAG biosynthesis in these lines being driven by the mass action of available acyl groups and not by altered specificity of the acyltransferases, the compounds responsible for incorporating fatty acids into TAG.     

Detection of foreign fat in milk fat from different continents by triacylglycerol analysis

The adulteration of bovine milk fat with foreign fat can be detected by the analysis of its triacylglycerol (TAG) composition in combination with TAG formulae to calculate S‐values. With genuine milk fats these S‐values must not exceed certain limits already laid down in German and European analytical standards since 1994. The present validity of the TAG formulae and corresponding S‐ranges was confirmed by analysis of 148 recently produced European milk fat samples comprising a wide variation in lipid composition. Additionally, representative milk fat collections from New Zealand (n = 31) and South Africa (n = 10) were analyzed for their TAG composition to check the applicability of the TAG method to non‐European dairy products. These samples were identified as pure and unadulterated as well. All the analyzed samples (n = 189) provided S‐values clearly within the admissible limits. Consequently, the composition of genuine milk fat from different continents still fits the S‐ranges formerly established for Europe, and the TAG method most likely can be applied globally. Limitations have to be considered with technologically processed milk fat or fat from low‐fat milk products such as skim milk or buttermilk.     

A new conjugated linoleic acid isomer, 7 trans, 9 cis-octadecadienoic acid, in cow milk, cheese, beef and human milk and adipose tissue

The identity of a previously unrecognized conjugated linoleic acid (CLA) isomer, 7 trans, 9 cis-octadecadienoic acid (18∶2) was confirmed in milk, cheese, beef, human milk, and human adipose tissue. The 7 trans, 9 cis-18∶2 isomer was resolved chromatographically as the methyl ester by silver ion-high-performance liquid chromatography (Ag⁺-HPLC); it eluted after the major 9 cis, 11 trans-18∶2 isomer (rumenic acid) in the natural products analyzed. In the biological matrices in-vestigated by Ag⁺-HPLC, the 7 trans, 9 cis-18∶2 peak was generally due to the most abundant minor CLA isomer, ranging in concentration from 3 to 16% of total CLA. By gas chromatography (GC) with long polar capillary columns, the methyl ester of 7 trans, 9 cis-18∶2 was shown to elute near the leading edge of the major 9 cis, 11 trans-18∶2 peak, while the 4,4-dimethyloxazoline (DMOX) derivative permitted partial resolution of these two CLA isomers. The DMOX derivative of this new CLA isomer was analyzed by gas chromatography-electron ionization mass spectrometry (GC-EIMS). The double bond positions were at Δ7 and Δ9 as indicated by the characteristic mass spectral fragment ions at m/z 168, 180, 194, and 206, and their allylic cleavages at m/z 154 and 234. The cis/trans double-bond configuration was established by GC-direct deposition-Fourier transform infrared as evidenced from the doublet at 988 and 949 cm⁻¹ and absorptions at 3020 and 3002 cm⁻¹. The 7 trans, 9 cis-18∶2 configuration was established by GC-EIMS for the DMOX derivative of the natural products examined, and by comparison to a similar product obtained from treatment of a mixture of methyl 8-hydroxy-and 11-hydroxyoctadec-9 cis enoates with BF₃, in methanol. Contribution number S010 from the Food Research Center, Guelph, Ontario, Canada.     

Conjugated Linoleic Acid Reduces Hepatic Steatosis and Restores Liver Triacylglycerol Secretion and the Fatty Acid Profile During Protein Repletion in Rats

Protein depletion is associated with hepatic steatosis and decreased circulating triacylglycerol (TAG). Since conjugated linoleic acid (CLA) increases lean body mass, protects against muscle catabolism, and modulates lipid metabolism, the aim of this work was to investigate the effects of CLA with two different amounts of dietary fat on the regulation of plasma and hepatic TAG concentration, and its possible connections with changes in fatty acid (FA) profile in plasma, liver and adipose tissue and hepatic oxidative status during protein repletion. Rats were fed a low protein diet (14 days) and then a protein repletion diet (30 days), supplemented or not with CLA, containing 7% (w/w) or 20% (w/w) of fat. Hepatic TAG secretion and removal by muscle and adipose tissue lipoprotein lipase, FA profile and liver oxidative status were evaluated. Protein depletion affected hepatic TAG secretion and peripheral removal, decreasing plasma and increasing liver TAG concentration, whereas protein repletion with CLA improved these abnormalities independently of the amount of dietary fat by increasing hepatic TAG secretion. This prevention in the absence of CLA was not observed. CLA was incorporated in plasma and tissues (adipose > liver > plasma, and c9,t11-CLA > t10,c12-CLA), accompanied by alterations in FA composition, mainly in adipose tissue. The hepatic oxidative stress was overcome by protein repletion. CLA had a beneficial impact on TAG metabolism in protein repleted animals, preventing hepatic steatosis through higher hepatic TAG secretion.     

Dietary Fat Does Not Overcome trans-10, cis-12 Conjugated Linoleic Acid Inhibition of Milk Fat Synthesis in Lactating mice

Trans-10, cis-12 conjugated linoleic acid (CLA) is a potent inhibitor of milk fat synthesis in the cow and similarly reduces milk fat in rodents. The objective of this study was to determine whether dietary fat can overcome CLA inhibition of milk fat concentration in lactating mice. Wild type C57Bl/6J mice (n = 31) were fed semipurified diets containing either low fat (LF; 4% fat) or high fat (HF; 23.6% fat) starting 4–6 days postpartum. Dietary fat was increased by inclusion of high oleic sunflower oil. After 2 days on the experimental diets, lactating dams were orally dosed with either water (control) or trans-10, cis-12 CLA (20 mg/day) for 5 days. CLA treatment decreased pup growth similarly in both HF and LF diets. Milk fat percent was increased over 16% by the HF diet and decreased over 12% by CLA, but there was no interaction of dietary fat and CLA. Both CLA and the HF diet reduced the proportion of short- and medium-chain fatty acids that originate from de novo synthesis, and there was no interaction of diet and CLA. CLA had no effect on the percent of preformed fatty acids, but the HF diet increased their abundance. Dietary fat and CLA both modified mammary expression of lipogenic enzymes and regulators, but no interactions were observed. In conclusion, CLA reduced milk fat concentration and litter growth, but these effects were not overcome by increased dietary fat from high oleic sunflower oil. CLA inhibition of milk fat in the mammary gland is not substrate dependent, and the mechanism is independent from dietary supply of oleic acid.     

Maternal supplementation with CLA decreases milk fat in humans

CLA refers to isomers of octadecadienoic acid with conjugated double bonds. The most abundant form of CLA (rumenic acid (RA): c9,t11-18∶2) is found in milk and beef fat. Further, CLA supplements containing RA and t10,c12−18∶2 are now available. Consumption of commercially produced CLA has been shown to decrease adipose accretion in growing laboratory and production animals and cause milk fat depression in cows. We tested the hypothesis that CLA supplementation would increase milk CLA concentration and decrease milk fat content in humans. Breastfeeding women (n=9) participated in this double-blind, placebo-controlled, crossover study divided into three periods: intervention l (5 d), washout (7 d), and intervention II (5 d). Women were randomized to treatment order. During each intervention period, women consumed 1.5 g of CLA supplement or placebo (olive oil) daily; during the washout period, no supplements were consumed. Milk was collected by complete breast expression on the final day of each period; milk output was estimated by 24-h weighing on the penultimate day of each intervention period. Milk RA and t10,c12−18∶2 concentrations were greater (P<0.05) during the CLA treatment period as compared to the placebo period. Milk fat content was significantly lower during the CLA treatment, as compared to the placebo treatment (P<0.05). Data indicate no effect of treatment on milk output. Therefore, it would be prudent that lactating women not consume commercially available CLA supplements at this time. This paper was published in part in Masters, N., McGuire, M.A., and McGuire, M.K. (1999) Conjugated Linoleic Acid Supplementation and Milk Fat Content in Humans, FASEB J. 13, A697.