A practical and fast isolation of 12 cyclolinopeptides (linusorbs) from flaxseed oil via preparative HPLC with phenyl-hexyl column.

Linusorbs, known as cyclolinopeptides, are a group of cyclic hydrophobic peptides derived from flaxseed oil with various health benefits. However, the current research efforts on both the biological activities and antioxidant capacities of linusorbs are limited because of existing issues with their purification and characterization. A practical method based on preparative HPLC for isolating 12 linusorbs simultaneously was developed and factors such as the solvent selection, gradient elution program, flow rate, loaded mass, and loading concentration, were optimized. The optimum conditions were an initial acetonitrile (ACN) to water ratio of 40%, final ACN ratio of 80%, eluting time of 21 min, a flow rate of 16 mL/min, sample load of 12.5 mg, and concentration of 80 mg/mL (in methanol). The 12 linusorbs obtained were verified using off-line MS/MS, recording purities of above 95.5%. The method could serve as a practical and fast isolation method enabling further investigation of minor linusorbs.

The Anti-Cancer Effect of Linusorb B3 from Flaxseed Oil through the Promotion of Apoptosis, Inhibition of Actin Polymerization, and Suppression of Src Activity in Glioblastoma Cells.

Linusorbs (LOs) are natural peptides found in flaxseed oil that exert various biological activities. Of LOs, LOB3 ([1-9-NαC]-linusorb B3) was reported to have antioxidative and anti-inflammatory activities; however, its anti-cancer activity has been poorly understood. Therefore, this study investigated the anti-cancer effect of LOB3 and its underlying mechanism in glioblastoma cells. LOB3 induced apoptosis and suppressed the proliferation of C6 cells by inhibiting the expression of anti-apoptotic genes, B cell lymphoma 2 (Bcl-2) and p53, as well as promoting the activation of pro-apoptotic caspases, caspase-3 and -9. LOB3 also retarded the migration of C6 cells, which was achieved by suppressing the formation of the actin cytoskeleton critical for the progression, invasion, and metastasis of cancer. Moreover, LOB3 inhibited the activation of the proto-oncogene, Src, and the downstream effector, signal transducer and activator of transcription 3 (STAT3), in C6 cells. Taken together, these results suggest that LOB3 plays an anti-cancer role by inducing apoptosis and inhibiting the migration of C6 cells through the regulation of apoptosis-related molecules, actin polymerization, and proto-oncogenes.

LOMIX, a Mixture of Flaxseed Linusorbs, Exerts Anti-Inflammatory Effects through Src and Syk in the NF-κB Pathway.

Although flax (Linum usitatissimum L.) has long been used as Ayurvedic medicine, its anti-inflammatory role is still unclear. Therefore, we aimed to investigate the anti-inflammatory role of a linusorb mixture (LOMIX) recovered from flaxseed oil. Effects of LOMIX on inflammation and its mechanism of action were examined using several in vitro assays (i.e., NO production, real-time PCR analysis, luciferase-reporter assay, Western blot analysis, and kinase assay) and in vivo analysis with animal inflammation models as well as acute toxicity test. Results: LOMIX inhibited NO production, cell shape change, and inflammatory gene expression in stimulated RAW264.7 cells through direct targeting of Src and Syk in the NF-κB pathway. In vivo study further showed that LOMIX alleviated symptoms of gastritis, colitis, and hepatitis in murine model systems. In accordance with in vitro results, the in vivo anti-inflammatory effects were mediated by inhibition of Src and Syk. LOMIX was neither cytotoxic nor did it cause acute toxicity in mice. In addition, it was found that LOB3, LOB2, and LOA2 are active components included in LOMIX, as assessed by NO assay. These in vitro and in vivo results suggest that LOMIX exerts an anti-inflammatory effect by inhibiting the inflammatory responses of macrophages and ameliorating symptoms of inflammatory diseases without acute toxicity and is a promising anti-inflammatory medication for inflammatory diseases.

Structural Modifications of a Flaxseed Lignan in Pursuit of Higher Liposolubility: Evaluation of the Antioxidant and Permeability Properties of the Resulting Derivatives.

While lignans and their biogenetic precursors can have various health benefits, the poor liposolubilities of such phenolic systems have restricted their application as antioxidants in the food industry. The research reported here was aimed at addressing these matters through derivatizing certain forms of such compounds and then assessing their properties as potential nutraceuticals. In particular, crude flaxseed lignan was purified to afford secoisolariciresinol diglucoside (SDG, 1) that was then subjected to structural modification. By such means, the SDG long-chain fatty acid esters 4-9 and 11-13, the fully acetylated SDG 10, secoisolariciresinol (SECO, 2), and anhydrosecoisolariciresinol (ASECO, 14) were obtained. The antioxidant activities of these derivatives were determined while their permeability properties were evaluated. Such studies revealed that certain SDG derivatives possessing useful liposolubilities also retained their antioxidative properties, as well as being capable of permeating Caco-2 cell monolayers while being nontoxic to them. SDG fatty acid esters 4-9 and 11-13 could be developed into emulsifiers with enhanced health benefits, especially considering their improved antioxidative (ca. <11 000 µmol Trolox/g) and permeability properties. This study thus highlights strategies for the structural modification of SDG so as to generate derivatives with superior properties in terms of their utility in the food and pharmaceutical industries.

Iron Fortification and Bioavailability of Chickpea (Cicer arietinum L.) Seeds and Flour.

Iron (Fe) deficiency is one of the most common nutritional disorders, and is mainly due to insufficient intake of bioavailable Fe. Chickpea (Cicer arietinum L.) was examined as a potential vehicle for Fe fortification. Fortificants (FeSO4·7H2O (ferrous sulfate hepta-hydrate), FeSO4·H2O (ferrous sulfate mono-hydrate) and NaFeEDTA (ethylenediaminetetraacetic acid iron (iii) sodium salt)) were applied by a spraying and drying method. At 2000 µg g-1 iron fortificant, the fortified split desi seeds (dal), desi flour and kabuli flour supplied 18-19 mg, 16-20 mg and 11-19 mg Fe per 100 g, respectively. The overall consumer acceptability using a nine-point hedonic scale for sensory evaluation demonstrated that NaFeEDTA-fortified cooked chickpea (soup and chapatti) scored the highest among the three fortificants. Lightness (L*), redness (a*) and yellowness (b*) of Fe-fortified products changed over time. However, no organoleptic changes occurred. Fe bioavailability was increased by 5.8-10.5, 15.3-25.0 and 4.8-9.0 ng ferritin mg-1 protein for cooked split desi seeds (soup), desi chapatti and kabuli chapatti, respectively, when prepared using Fe-fortified chickpea. Desi chapatti showed significantly higher Fe bioavailability than the other two. The increase in Fe concentration and bioavailability in fortified chickpea products demonstrated that these products could provide a significant proportion of the recommended daily Fe requirement.

Novel Flaxseed Gum Nanocomposites Are Slow Release Iron Supplements.

Nanocomposites, based on iron salts and soluble flaxseed gum (FG), were prepared as potential treatments of iron deficiency anemia (IDA). FG was extracted, characterized, and formulated into iron-loading nanocomposites via ion-exchange against FeCl3, Fe2(SO4)3, FeCl2, and FeSO4·7H2O. FG-iron nanocomposites preparation condition was optimized, and physicochemical properties of the nanocomposites were investigated. In vitro release kinetics of iron in simulated gastric fluid (SGF) was also evaluated. FG heteropolysaccharide, consisting of rhamnose (33.73%), arabinose (24.35%), xylose (14.23%), glucose (4.54%), and galactose (23.15%) monosaccharides, linked together via varieties of glycosidic bonds, was a good recipient for both ferric and ferrous irons under screened conditions (i.e., 80 °C, 2 h, I/G = 1:2). Iron loaded contents in the nanocomposites prepared from FG-FeCl3, FG-Fe2(SO4)3, FG-FeCl2, and FG-FeSO4·7H2O were 25.51%, 10.36%, 5.83%, and 22.83%, respectively. Iron in these nanocomposites was mostly in a bound state, especially in FG-FeCl3, due to chelation forming bonds between iron and polysaccharide hydroxyl or carboxyl groups and formed stable polysaccharide-iron crystal network structures. Free iron ions were effectively removed by ethanol treatments. Because of chelation, the nanocomposites delayed iron release in SGF and the release kinetics were consistent with Korsmeyer-Peppas model. This indicates that such complexes might reduce side effects of free iron in human stomach. Altogether, this study indicates that these synthetic FG-iron nanocomposites might be developed as novel iron supplements for iron deficiency, in which FG-FeCl3 is considered as the best option.

Secoisolariciresinol Diglucoside and Cyanogenic Glycosides in Gluten-free Bread Fortified with Flaxseed Meal.

Flaxseed (Linum usitatissimum L.) meal contains cyanogenic glycosides (CGs) and the lignan secoisolariciresinol diglucoside (1). Gluten-free (GF) doughs and baked goods were produced with added flaxseed meal (20%, w/w) then 1, and CGs were determined in fortified flour, dough, and bread with storage (0, 1, 2, and 4 weeks) at different temperatures (-18, 4, and 22-23 °C). 1 was present in flour, dough, and GF bread after baking. 1 was stable with extensive storage (up to 4 weeks) and was not affected by storage temperature. CGs in flaxseed meal and fortified GF samples were analyzed by 1H NMR of the cyanohydrins. Linamarin and/or linustatin were the primary CGs in both flaxseed meal and fortified flour. CGs decreased with storage in dough fortified with flaxseed meal or GF bread after baking. GF bakery food products fortified with flaxseed meal had reduced CGs but remained a good source of dietary 1.

Vaccenic acid suppresses intestinal inflammation by increasing anandamide and related N-acylethanolamines in the JCR:LA-cp rat.

Vaccenic acid (VA), the predominant ruminant-derivedtransfat in the food chain, ameliorates hyperlipidemia, yet mechanisms remain elusive. We investigated whether VA could influence tissue endocannabinoids (ECs) by altering the availability of their biosynthetic precursor, arachidonic acid (AA), in membrane phospholipids (PLs). JCR:LA-cprats were assigned to a control diet with or without VA (1% w/w),cis-9,trans-11 conjugated linoleic acid (CLA) (1% w/w) or VA+CLA (1% + 0.5% w/w) for 8 weeks. VA reduced the EC, 2-arachidonoylglycerol (2-AG), in the liver and visceral adipose tissue (VAT) relative to control diet (P< 0.001), but did not change AA in tissue PLs. There was no additive effect of combining VA+CLA on 2-AG relative to VA alone (P> 0.05). Interestingly, VA increased jejunal concentrations of anandamide and those of the noncannabinoid signaling molecules, oleoylethanolamide and palmitoylethanolamide, relative to control diet (P< 0.05). This was consistent with a lower jejunal protein abundance (but not activity) of their degrading enzyme, fatty acid amide hydrolase, as well as the mRNA expression of TNFα and interleukin 1ß (P< 0.05). The ability of VA to reduce 2-AG in the liver and VAT provides a potential mechanistic explanation to alleviate ectopic lipid accumulation. The opposing regulation of ECs and other noncannabinoid lipid signaling molecules by VA suggests an activation of benefit via the EC system in the intestine.

Proposed systematic nomenclature for orbitides.

Orbitides are short (5-11 amino acid residue), ribosomally synthesized homodetic plant cyclic peptides characterized by N-to-C amide bonds rather than disulfide bonds. Orbitides can be discovered using mass spectrometry of plant extracts or by identifying DNA sequences coding for the precursor protein. The number of orbitides that have been characterized to date, by a number of different research groups, is modest. The nomenclatural system currently used for the Type VI cyclic peptides has been developed in an ad hoc fashion and is somewhat arbitrary. We propose a systematic naming system specifically for the Type VI cyclic peptides that reflects the taxonomic name of the species producing the orbitides and a numbering system that enables systematic representation of amino acid residues and modifications. The proposed naming system emulates the IUPAC Nomenclature for Natural Products and UniProt, both of which use abbreviations of taxonomic names for the compounds in question. Nomenclature for post-translational modifications also follows the IUPAC precedent, as well as the cyclic peptide literature. Furthermore, the proposed system aims to maintain agreement with the precedents set by the pre-existing literature. An example of the proposed nomenclature is provided using the methionine-containing homodetic peptides of Linum usitatissimum (flaxseed).

Glycine-containing flaxseed orbitides.

Five new orbitides, cyclolinopeptides 21-25, were identified in flaxseed oil (Linum usitatissimum) extracts. Their HPLC-ESIMS quasimolecular ion peaks at m/z 1097.7 (21), 1115.6 (22), 1131.6 (23), 1018.6 (24), and 1034.6 (25) [M + H](+) corresponded to the molecular formulae C59H89N10O10, C58H87N10O10S, C58H87N10O11S, C53H80N9O9S, and C53H80N9O10S, respectively. Their structures were elucidated by extensive HPLC-ESIMS/MS analyses, and their presence was confirmed by precursor proteins identified in flax genomic DNA sequence data. The amino acid sequences of these orbitides were confirmed as [1-10-NαC]-GILVPPFFLI, [1-10-NαC]-GMLIPPFFVI, [1-10-NαC]-GOLIPPFFVI, [1-9-NαC]-GMLVFPLFI, and [1-9-NαC]-GOLVFPLFI for cyclolinopeptides 21-25, respectively. Previously reported orbitides, [1-9-NαC]-ILVPPFFLI (1), [1-9-NαC]-MLIPPFFVI (2), [1-9-NαC]-OLIPPFFVI (3), [1-8-NαC]-MLVFPLFI (7), and [1-8-NαC]-OLVFPLFI (8), were also present in flaxseed oil. The precursors of orbitides 21, 22, and 24 also produced orbitides 1, 2, and 7 by alternative cyclization. Cyclolinopeptides 3, 8, 23, and 25 contain MetO (O) and are not directly encoded, but are products of post-translational modification of the Met present in 2, 7, 22, and 24, respectively. Sufficient cyclolinopeptide 23 was isolated for characterization via 1D ((1)H and (13)C) and 2D (NOESY and HMBC) NMR spectroscopy. These compounds have been named as cyclolinopeptides U, V, W, X, and Y for 21, 22, 23, 24, and 25, respectively.

Simulated moving bed purification of flaxseed oil orbitides: unprecedented separation of cyclolinopeptides C and E.

The purification and enrichment of most natural products with potential pharmaceutical applications has been performed mainly employing conventional batch-mode chromatographic processes. There is a growing interest in use of simulated moving bed (SMB) chromatography for natural product enrichment as this method enables conservation of mobile phase, while increasing productivity of chromatography medium. SMB increases yield while decreasing cost. Cyclolinopeptides C ([1-9-NaC],[1-MetO]-CLB, 3) and E ([1-8-NaC],[1-MetO]-CLE, 8) were extracted as a mixture from flaxseed oil and then enriched using a three-zone simulated moving bed. The current research extends the SMB technology to enrichment of cyclolinopeptides (CLs), a group of biologically active hydrophobic cyclic peptides that occur in flaxseed oil. Of interest are [1-9-NaC],[1-MetO]-CLB (3) and [1-8-NaC],[1-MetO]-CLE (8) that provide synthetic scaffolds for modified CLs. The influence of flow rate (feed, desorbent, and extract) on the separation of [1-9-NaC],[1-MetO]-CLB (3) and [1-8-NaC],[1-MetO]-CLE (8) was investigated.

Diets enriched in trans-11 vaccenic acid alleviate ectopic lipid accumulation in a rat model of NAFLD and metabolic syndrome.

Trans11-18:1 (vaccenic acid, VA) is one of the most predominant naturally occurring trans fats in our food chain and has recently been shown to exert hypolipidemic effects in animal models. In this study, we reveal new mechanism(s) by which VA can alter body fat distribution, energy utilization and dysfunctional lipid metabolism in an animal model of obesity displaying features of the metabolic syndrome (MetS). Obese JCR:LA-cp rats were assigned to a control diet that included dairy-derived fat or the control diet supplemented with 1% VA. VA reduced total body fat (-6%), stimulated adipose tissue redistribution [reduced mesenteric fat (-17%) while increasing inguinal fat mass (29%)] and decreased adipocyte size (-44%) versus control rats. VA supplementation also increased metabolic rate (7%) concomitantly with an increased preference for whole-body glucose utilization for oxidation and increased insulin sensitivity [lower HOMA-IR (-59%)]. Further, VA decreased nonalcoholic fatty liver disease activity scores (-34%) and reduced hepatic (-27%) and intestinal (-39%) triglyceride secretion relative to control diet, while exerting differential transcriptional regulation of SREBP1 and FAS amongst other key genes in the liver and the intestine. Adding VA to dairy fat alleviates features of MetS potentially by remodeling adipose tissue and attenuating ectopic lipid accumulation in a rat model of obesity and MetS. Increasing VA content in the diet (naturally or by fortification) may be a useful approach to maximize the health value of dairy-derived fats.

Effect of cyclolinopeptides on the oxidative stability of flaxseed oil.

Polar compounds present in flaxseed oil increase its oxidative stability. Flaxseed oil becomes less stable to oxidation when filtered with silica. This observation may be linked to antioxidant compounds present in flaxseed oil. Flaxseed oil was passed over a silica adsorbent column to remove polar compounds. The polar compounds were then eluted from the silica absorbant using a series of increasingly polar solvents. The polar fractions from flaxseed oil were then added back to silica-treated flaxseed oil to determine the impact of fractions containing polar compounds on oxidative stability (induction time) at 100 °C. A polar fraction containing mainly cyclolinopeptide A (CLA, 1), but also containing ß/γ- and δ-tocopherol increased the induction time of silica-treated flaxseed oil from 2.36 ± 0.28 to 3.20 ± 0.41 h. When oxidative stability was determined immediately after addition of the polar fractions other flaxseed fractions and solvent controls did not affect oil stability. However, when the oxidative stability index (OSI) test was delayed for three days and oil samples were held at room temperature after the addition of the polar fractions to the flaxseed oil, it was observed that the control oil treated with silica had become highly sensitive to oxidation. A polar fraction containing a mixture of CLs (1, 5, 7, 9, 11), improved the oxidative stability of peptide-free oil with respect to the control when the OSI measurement was made three days after adding the fraction. In addition, effects of 1 on the oxidative stability of peptide-free oil containing divalent metal cations was investigated.

Identification and quantification of cyclolinopeptides in five flaxseed cultivars.

Cyclolinopeptides are a group of naturally occurring hydrophobic cyclic peptides found in flaxseed and flax oil that have immunosuppressive activity. This study describes the measurement of flaxseed cyclolinopeptide concentrations using an internal standard HPLC method. In addition, the concentration of cyclolinopeptides in the seed of Canadian flax cultivars grown at two locations over two years is reported. The data are consistent with the formation of flaxseed cyclolinopeptides from two ribosome-derived precursors. Each precursor protein includes the sequences corresponding to three cyclolinopeptides from which those cyclolinopeptides are presumably derived by precursor processing. The concentrations of cyclolinopeptides C and E, which are encoded by the same gene sequence, are highly correlated, and the concentrations of cyclolinopeptides D, F, and G, which are encoded by a second gene sequence, are also highly correlated. The strong correlation between the cyclolinopeptides arising from the same gene may prove to be important in understanding how peptide concentration is controlled. Additional research may lead to approaches to improve flax either as a platform for peptide production or as a source of oil with improved drying properties and flavor.

Distribution of cyclolinopeptides in flaxseed fractions and products.

Hydrophobic cyclic peptides, termed cyclolinopeptides, found in flaxseed are known for their immunosuppressive activity. This study is the first report of the occurrence of cyclolinopeptides in flaxseed fractions and products produced by aqueous processing and cold pressing. The distribution of cyclolinopeptides in flaxseed was determined after processing of flaxseed by various industrial and laboratory processes. Extracts of the water-soluble mucilage did not contain cyclolinopeptides. The cotyledon had the highest concentration of cyclolinopeptides, whereas seed coat had lower levels. An oil body fraction separated from seed after homogenization in water, followed by centrifugation, had the highest concentration of cyclolinopeptides of the fractions produced by this method. Further washing of the oil body fraction led to a loss of cyclolinopeptides. When oilseed was extruded using an expeller press, cyclolinopeptides were found in greater concentrations in crude oil and the solid sediment present in the oil fraction than in meal or the unprocessed seed. The concentration of cyclolinopeptides in crude flaxseed oil immediately after pressing was much higher than that observed in flaxseed oils purchased from a retail outlet. The effect of oil refining treatments on the removal of cyclolinopeptides was also tested. Acid degumming using aqueous H(3)PO(4) removed cyclolinopeptides from crude flaxseed oil. Alkali refining was less effective as this treatment failed to remove all peptides equally. This work illustrates ways that cyclolinopeptides may be extracted from flaxseed oil that could be developed for large-scale industrial extraction. The ability to extract cyclolinopeptides on a larger scale would allow faster exploitation of commercial applications of these molecules and provide the flaxseed industry with value-added coproducts.

Rapid reversed-phase liquid chromatography separation of cyclolinopeptides with monolithic and microparticulate columns.

Three monolithic C(18)-bonded silica gel columns i.e. Chromolith SpeedROD (CSR), Chromolith Performance (CP), and Chromolith High Resolution (CHR), MerckKGaA Darmstadt, Germany and two particle-based columns i.e. ZORBAX Eclipse XDB-C(18) (ZEX), Agilent and POROS R1/20 (POR), Applied Biosystems were compared for their performance in separating a mixture of flaxseed cyclolinopeptides (CLs). Gradient mobile phases of acetonitrile and water were optimized for each column. The performance of CHR column in profiling CL standards, measured as the resolution of individual CL, selectivity, and peak asymmetry exceeded the performance of traditional particle-packed columns and the other monolithic columns. The profiling of CLs in aqueous methanolic flaxseed extract was optimized for high-throughput analysis. A total analysis time of 1.5 min at a flow rate of 3.0mLmin(-1) was achieved on a CSR column. Injection of over 2000 methanol extracts of flaxseed on a CSR column had no impact on backpressure or resolution of a standard CL mixture.

Rapid NMR method for the quantification of organic compounds in thin stillage.

Thin stillage contains organic and inorganic compounds, some of which may be valuable fermentation coproducts. This study describes a thorough analysis of the major solutes present in thin stillage as revealed by NMR and HPLC. The concentration of charged and neutral organic compounds in thin stillage was determined by excitation sculpting NMR methods (double pulse field gradient spin echo). Compounds identified by NMR included isopropanol, ethanol, lactic acid, 1,3-propanediol, acetic acid, succinic acid, glycerophosphorylcholine, betaine, glycerol, and 2-phenylethanol. The concentrations of lactic and acetic acid determined with NMR were comparable to those determined using HPLC. HPLC and NMR were complementary, as more compounds were identified using both methods. NMR analysis revealed that stillage contained the nitrogenous organic compounds betaine and glycerophosphorylcholine, which contributed as much as 24% of the nitrogen present in the stillage. These compounds were not observed by HPLC analysis.

The biosynthesis of Caryophyllaceae-like cyclic peptides in Saponaria vaccaria L. from DNA-encoded precursors.

Cyclic peptides (CPs) are produced in a very wide range of taxa. Their biosynthesis generally involves either non-ribosomal peptide synthases or ribosome-dependent production of precursor peptides. Plants within the Caryophyllaceae and certain other families produce CPs which generally consist of 5-9 proteinogenic amino acids. The biological roles for these CPs in the plant are not very clear, but many of them have activity in mammalian systems. There is currently very little known about the biosynthesis of CPs in the Caryophyllaceae. A collection of expressed sequence tags from developing seeds of Saponaria vaccaria was investigated for information about CP biosynthesis. This revealed genes that appeared to encode CP precursors which are subsequently cyclized to mature CPs. This was tested and confirmed by the expression of a cDNA encoding a putative precursor of the CP segetalin A in transformed S. vaccaria roots. Similarly, extracts of developing S. vaccaria seeds were shown to catalyze the production of segetalin A from the same putative (synthetic) precursor. Moreover, the presence in S. vaccaria seeds of two segetalins, J [cyclo(FGTHGLPAP)] and K [cyclo(GRVKA)], which was predicted by sequence analysis, was confirmed by liquid chromatography/mass spectrometry. Sequence analysis also predicts the presence of similar CP precursor genes in Dianthus caryophyllus and Citrus spp. The data support the ribosome-dependent biosynthesis of Caryophyllaceae-like CPs in the Caryophyllaceae and Rutaceae.

Increased hypolipidemic benefits of cis-9, trans-11 conjugated linoleic acid in combination with trans-11 vaccenic acid in a rodent model of the metabolic syndrome, the JCR:LA-cp rat.

BACKGROUND: Conjugated linoleic acid (cis-9, trans-11 CLA) and trans-11 vaccenic acid (VA) are found naturally in ruminant-derived foods. CLA has been shown to have numerous potential health related effects and has been extensively investigated. More recently, we have shown that VA has lipid-lowering properties associated with reduced hepatic lipidogenesis and chylomicron secretion in the JCR:LA-cp rat. The aim of this study was to evaluate potential additional hypolipidemic effects of purified forms of CLA and VA in an animal model of the metabolic syndrome (the JCR:LA-cp rat). METHODS: Twenty four obese JCR:LA-cp rats were randomized and assigned to one of three nutritionally adequate iso-caloric diets containing 1% w/w cholesterol and 15% w/w fat for 16 wk: 1) control diet (CD), 2) 1.0% w/w cis-9, trans-11 CLA (CLA), 3) 1.0% w/w VA and 1% w/w cis-9, trans-11 CLA (VA+CLA). Lean rats were fed the CD to represent normolipidemic conditions. RESULTS: Fasting plasma triglyceride (TG), total cholesterol and LDL-cholesterol concentrations were reduced in obese rats fed either the CLA diet or the VA+CLA diet as compared to the obese control group (p < 0.05, p < 0.001; p < 0.001, p < 0.01; p < 0.01, p < 0.001, respectively). The VA+CLA diet reduced plasma TG and LDL-cholesterol to the level of the normolipidemic lean rats and further decreased nonesterified fatty acids compared to the CLA diet alone. Interestingly, rats fed the VA+CLA diet had a higher food intake but lower body weight than the CLA fed group (P < 0.05). Liver weight and TG content were lower in rats fed either CLA (p < 0.05) or VA+CLA diets (p < 0.001) compared to obese control, consistent with a decreased relative protein abundance of hepatic acetyl-CoA carboxylase in both treatment groups (P < 0.01). The activity of citrate synthase was increased in liver and adipose tissue of rats fed, CLA and VA+CLA diets (p < 0.001) compared to obese control, suggesting increased mitochondrial fatty acid oxidative capacity. CONCLUSION: We demonstrate that the hypolipidemic effects of chronic cis-9, trans-11 CLA supplementation on circulating dyslipidemia and hepatic steatosis are enhanced by the addition of VA in the JCR:LA-cp rat.

Trans-11 vaccenic acid reduces hepatic lipogenesis and chylomicron secretion in JCR:LA-cp rats.

Trans-11 vaccenic acid (VA) is the predominant trans isomer in ruminant fat and a major precursor to the endogenous synthesis of cis9,trans11-conjugated linoleic acid in humans and animals. We have previously shown that 3-wk VA supplementation has a triglyceride (TG)-lowering effect in a rat model of dyslipidemia, obesity, and metabolic syndrome (JCR:LA-cp rats). The objective of this study was to assess the chronic effect (16 wk) of VA on lipid homeostasis in both the liver and intestine in obese JCR:LA-cp rats. Plasma TG (P < 0.001), total cholesterol (P < 0.001), LDL cholesterol (P < 0.01), and nonesterified fatty acid concentrations, as well as the serum haptoglobin concentration, were all lower in obese rats fed the VA diet compared with obese controls (P < 0.05). In addition, there was a decrease in the postprandial plasma apolipoprotein (apo)B48 area under the curve (P < 0.05) for VA-treated obese rats compared with obese controls. The hepatic TG concentration and the relative abundance of fatty acid synthase and acetyl-CoA carboxylase proteins were all lower (P < 0.05) in the VA-treated group compared with obese controls. Following acute gastrointestinal infusion of a VA-triolein emulsion in obese rats that had been fed the control diet for 3 wk, the TG concentration was reduced by 40% (P < 0.05) and the number of chylomicron (CM) particles (apoB48) in nascent mesenteric lymph was reduced by 30% (P < 0.01) relative to rats infused with a triolein emulsion alone. In conclusion, chronic VA supplementation significantly improved dyslipidemia in both the food-deprived and postprandial state in JCR:LA-cp rats. The appreciable hypolipidemic benefits of VA may be attributed to a reduction in both intestinal CM and hepatic de novo lipogenesis pathways.