Fatty acid concentration of plasma, muscle, adipose and liver from beef heifers fed an encapsulated n-3 polyunsaturated fatty acid supplement.

Increasing the content of polyunsaturated fat in the human diet is a priority for reducing cardiovascular disease and cancer risks. Beef has the potential to contribute to the polyunsaturated fat content in the human diet; however, ruminants cannot synthesise many long-chain fatty acids de novo; they require dietary supplementation. The objectives of the current study were to evaluate (i) the effect of a partially rumen protected n-3 long-chain polyunsaturated fatty acid (LC-PUFA) dietary supplement on the fatty acid composition of muscle (Longissimus dorsi), adipose and liver tissues of beef heifers and (ii) the usefulness of blood plasma as a predictor of tissue concentrations of specific fatty acids. Charolais crossbred heifers (n = 20) were assigned to one of two isolipid dietary treatments namely palmitic acid (control) or an n-3 LC-PUFA supplement for a 91-day period. Blood plasma and adipose tissue samples were taken to determine the temporal effect of these diets on fatty acid composition (days 0, 10, 35 and 91), while liver and muscle samples were taken following slaughter. Dietary lipid source did not influence animal growth rate or body condition score. At day 91, the percentage differences between control and n-3 LC-PUFA heifers in concentrations of eicosapentaenoic acid were +61, +176 and +133 % in liver, muscle and adipose, respectively. For docosahexaenoic acid, at the same time point, the percentage differences were +57, +73 and +138 % for liver, muscle and adipose, respectively. Medium-to-strong positive correlation coefficients were evident for liver and plasma fatty acids, in particular, there were positive relationships with concentrations of total saturated fatty acid (SFA), total n-6 PUFA and total n-3 PUFA. This trend also extended to both the ratio of PUFA to SFA (slope (ß1) = 0.56 ±â€¯0.167, intercept (ß0) = 0.56, R2 = 0.61, P < 0.05) and the ratio of n-6 to n-3 PUFA (ß1 = 0.15 ±â€¯0.054, ß0 = 0.24, R2 = 0.52, P < 0.05). A strong correlation was also detected in the ratio of n-6 to n-3 in plasma and muscle tissue of heifers fed the n-3 LC-PUFA diet (ß1 = 0.53 ±â€¯0.089, ß0 = -0.31, R2 = 0.83, P < 0.001). The results of this study show that the n-3 LC-PUFA can be readily increased through targeted supplementation and that plasma concentrations of n-3 LC-PUFA are useful predictors of their concentrations in a number of economically important tissues.

Optimization of a reconstituted skim milk based medium for enhanced CLA production by bifidobacteria.

AIMS: To determine the effect of a range of supplements on the bioconversion of linoleic acid to conjugated linoleic acid (CLA) by Bifidobacterium breve NCIMB 702258 in reconstituted skim milk (RSM). RESULTS: Seven supplements (yeast extract, casein hydrolysate, tryptone, l-cysteine hydrochloride, sodium acetate, sodium butyrate and sodium propionate) were identified as increasing the bioconversion of linoleic acid to c9, t11 CLA. Using these supplements, the percentage bioconversion of linoleic acid (0.35 mg ml(-l)) to the c9, t11 CLA isomer was elevated from 15.5 +/- 1.1% in 20% RSM (w/v) to 48.1 +/- 2.2% in the supplemented RSM. Through additional supplementation of 20 mg m1(-1) inulin and optimization of inoculum and linoleic acid concentration, the percentage bioconversion to c9, t11 CLA was increased to 55.0 + 3.2%. CONCLUSIONS: Through supplementation, the concentration of CLA produced by bifidobacteria in RSM can be increased to levels comparable to those observed in the synthetic medium cys-MRS. SIGNIFICANCE AND IMPACT OF THE STUDY: The impact of 22 supplements on the production of the c9, t11 CLA isomer by the strain B. breve NCIMB 702258 in milk has been determined. The results provide an understanding of the factors, which influence CLA production by bifidobacteria in RSM.

Cheese manufacture with milk with elevated conjugated linoleic acid levels caused by dietary manipulation.

The objective of this study was to assess the effect of dietary supplementation of cows on pasture with sunflower oil for conjugated linoleic acid (cis-9, trans-11 CLA) enrichment of milk, for the production of CLA-enriched cheese. A group of 40 autumn-calving dairy cows were assigned to either a control group (indoor feeding on grass silage ad libitum and 6 kg/d of a typical indoor concentrate) or an experimental group (on pasture, being fed 6 kg of a supplement containing 100 g/kg of sunflower oil per d). These diets were fed for 16 d, during which time milk was collected for pilot-scale hard cheese manufacture. The pasture-based diet with sunflower oil resulted in a significant effect on the milk fatty acid CLA content. The concentration of cis-9, trans-11 CLA in the milk produced from cows on this diet increased to 2.22 g/100 g of fatty acid methyl esters (FAME) after 14 d, compared with 0.46 g/100 g of FAME in milk produced on the control indoor diet. The content of cis-9, trans-11 CLA in the cheese manufactured from the indoor control milk was 0.78 g/100 g of FAME and that from the pasture-based sunflower oil milk was 1.93 g/100 g of FAME. The cheese was assessed during the ripening period and CLA concentrations were stable throughout the 6 mo of ripening. Other cheese variables (microbiology, composition, flavor, free AA) were monitored during the ripening period, and the cheese with the elevated CLA concentrations compared favorably with the control cheese. Thus, a pasture-based diet supplemented with an oil source rich in linoleic acid resulted in an enhanced CLA content of bovine milk fat, compared with an indoor grass silage-based diet.

Mining the microbiota of the neonatal gastrointestinal tract for conjugated linoleic acid-producing bifidobacteria.

This study was designed to isolate different strains of the genus Bifidobacterium from the fecal material of neonates and to assess their ability to produce the cis-9, trans-11 conjugated linoleic acid (CLA) isomer from free linoleic acid. Fecal material was collected from 24 neonates aged between 3 days and 2 months in a neonatal unit (Erinville Hospital, Cork, Ireland). A total of 46 isolates from six neonates were confirmed to be Bifidobacterium species based on a combination of the fructose-6-phosphate phosphoketolase assay, RAPD [random(ly) amplified polymorphic DNA] PCR, pulsed-field gel electrophoresis (PFGE), and partial 16S ribosomal DNA sequencing. Interestingly, only 1 of the 11 neonates that had received antibiotic treatment produced bifidobacteria. PFGE after genomic digestion with the restriction enzyme XbaI demonstrated that the bifidobacteria population displayed considerable genomic diversity among the neonates, with each containing between one and five dominant strains, whereas 11 different macro restriction patterns were obtained. In only one case did a single strain appear in two neonates. All genetically distinct strains were then screened for CLA production after 72 h of incubation with 0.5 mg of free linoleic acid ml(-1) by using gas-liquid chromatography. The most efficient producers belonged to the species Bifidobacterium breve, of which two different strains converted 29 and 27% of the free linoleic acid to the cis-9, trans-11 isomer per microgram of dry cells, respectively. In addition, a strain of Bifidobacterium bifidum showed a conversion rate of 18%/microg dry cells. The ability of some Bifidobacterium strains to produce CLA could be another human health-promoting property linked to members of the genus, given that this metabolite has demonstrated anticarcinogenic activity in vitro and in vivo.

Conjugated linoleic acid biosynthesis by human-derived Bifidobacterium species.

AIMS: To assess strains of Lactobacillus, Lactococcus, Pediococcus and Bifidobacterium for their ability to produce the health-promoting fatty acid conjugated linoleic acid (CLA) from free linoleic acid. METHODS AND RESULTS: In this study, strains of Lactobacillus, Lactococcus, Pediococcus and Bifidobacterium were grown in medium containing free linoleic acid. Growth of the bacteria in linoleic acid and conversion of the linoleic acid to CLA was assessed. Of the bacteria assessed, nine strains of Bifidobacterium produced the c9, t11 CLA isomer from free linoleic acid. The t9, t11 CLA isomer was also produced by some strains, but at much lower concentrations. CONCLUSIONS: The production of CLA by bifidobacteria exhibited considerable interspecies variation. Bifidobacterium breve and B. dentium were the most efficient CLA producers among the range of strains tested, with B. breve converting up to 65% linoleic acid to c9, t11 CLA when grown in 0.55 mg ml(-1) linoleic acid. Strains also varied considerably with respect to their sensitivity to linoleic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of CLA by probiotic bifidobacteria offers a possible mechanism for some health-enhancing properties of bifidobacteria and provides novel opportunities for the development of functional foods.

Conjugated linoleic acid and oxidative behaviour in cancer cells.

Convincing evidence from rodent models of carcinogenesis indicates that cis-9,trans-11 (c9t11) conjugated linoleic acid (CLA) is a potent naturally occurring anti-carcinogen in the human diet. CLA has been reported to alter the fatty acid composition of biological tissues in a manner that increases their oxidative stability. However, recent information suggests that an antioxidant role for CLA does not seem plausible. Given the knowledge that c9t11 CLA is present in a wide range of meat and dairy food products, our studies have begun to investigate mechanisms by which CLA-enriched milk fat exerts its anti-carcinogenic effects. An oxidative mechanism appears to be involved in its growth-suppressive effects, since supplementation of growth culture medium with CLA (17-71.5 microM) made breast cancer cells more susceptible to lipid peroxidation. Studies have indicated that cancer cells may become enriched in CLA during growth in culture. This may make intracellular lipids more susceptible to ordinary levels of oxidative stress, to the point of producing a cytotoxic effect.

Modulation of arachidonic acid distribution by conjugated linoleic acid isomers and linoleic acid in MCF-7 and SW480 cancer cells.

The relationship between growth and alterations in arachidonic acid (AA) metabolism in human breast (MCF-7) and colon (SW480) cancer cells was studied. Four different fatty acid preparations were evaluated: a mixture of conjugated linoleic acid (CLA) isomers (c9,t11, t10,c12, c11,t13, and minor amounts of other isomers), the pure c9,t11-CLA isomer, the pure t10,c12-CLA isomer, and linoleic acid (LA) (all at a lipid concentration of 16 microg/mL). 14C-AA uptake into the monoglyceride fraction of MCF-7 cells was significantly increased following 24 h incubation with the CLA mixture (P < 0.05) and c9,t11-CLA (P < 0.02). In contrast to the MCF-7 cells, 14C-AA uptake into the triglyceride fraction of the SW480 cells was increased while uptake into the phospholipids was reduced following treatment with the CLA mixture (P < 0.02) and c9,t11-CLA (P < 0.05). Distribution of 14C-AA among phospholipid classes was altered by CLA treatments in both cell lines. The c9,t11-CLA isomer decreased (P < 0.05) uptake of 14C-AA into phosphatidylcholine while increasing (P < 0.05) uptake into phosphatidylethanolamine in both cell lines. Both the CLA mixture and the t10,c12-CLA isomer increased (P < 0.01) uptake of 14C-AA into phosphatidylserine in the SW480 cells but had no effect on this phospholipid in the MCF-7 cells. Release of 14C-AA derivatives was not altered by CLA treatments but was increased (P < 0.05) by LA in the SW480 cell line. The CLA mixture of isomers and c9,t11-CLA isomer inhibited 14C-AA conversion to 14C-prostaglandin E2 (PGE2) by 20-30% (P < 0.05) while increasing 14C-PGF2alpha by 17-44% relative to controls in both cell lines. LA significantly (P < 0.05) increased 14C-PGD2 by 13-19% in both cell lines and increased 14C-PGE2 by 20% in the SW480 cell line only. LA significantly (P < 0.05) increased 5-hydroperoxyeicosatetraenoate by 27% in the MCF-7 cell line. Lipid peroxidation, as determined by increased levels of 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha), was observed following treatment with c9,t11-CLA isomer in both cell lines (P < 0.02) and with t10,c12-CLA isomer in the MCF-7 cell line only (P < 0.05). These data indicate that the growth-promoting effects of LA in the SW480 cell line may be associated with enhanced conversion of AA to PGE2 but that the growth-suppressing effects of CLA isomers in both cell lines may be due to changes in AA distribution among cellular lipids and an altered prostaglandin profile.

Milk fat conjugated linoleic acid (CLA) inhibits growth of human mammary MCF-7 cancer cells.

The relationship between growth and the antioxidant enzyme defence system in human MCF-7 (breast) cancer cells treated with bovine milk fat enriched with conjugated linoleic acid (CLA) was studied. Milk enriched in CLA was obtained from cows on pasture supplemented with full fat rapeseeds and full fat soyabeans (1). Cell number decreased up to 90% (p < 0.05) and lipid peroxidation increased 15-fold (p < 0.05) following incubation of MCF-7 cells for 8 days with increasing levels of milk fat yielding CLA concentrations between 16.9 and 22.6 ppm. Growth suppression and prooxidant effects of milk fat CLA were independent of the variable composition of the milk fat samples, suggesting that CLA was the active ingredient in milk fat responsible for the cytotoxic effect. Mixtures containing isomers of CLA (c9, t11-, t10, c12-, c11, t13- and minor amounts of other isomers) and linoleic acid (LA) at similar concentrations to the milk fat samples were as effective at inhibiting growth and stimulating peroxidation of MCF-7 cells as the milk fatty acids. Incubation of the cells with the c9, t11 CLA isomer (20 ppm) or the mixture of CLA isomers (20 ppm) for 8 days resulted in a 60% decrease (p < 0.05) in viability compared with untreated controls and was significantly (p < 0.05) more effective than incubation with the t10, c12 CLA isomer (20 ppm), which caused only a 15% decrease in cell numbers under similar conditions. A 25% increase (p < 0.05) in cell proliferation occurred when LA (20 ppm) alone was incubated with MCF-7 cells for 8 days. 14C-CLA was preferentially incorporated into the phospholipid fraction of the MCF-7 cell lipids in a dose-dependent manner and CLA accumulated in cell membranes more efficiently when the cells were incubated in the presence of milk fat than the c9, t11 synthetic CLA isomer. Superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activities were induced in MCF-7 cells exposed to milk fat (containing 16.9-22.6 ppm CLA) over 8 days. The data indicate that milk fat triglyceride-bound CLA, consisting primarily of the c9, t11 isomer, was cytotoxic towards MCF-7 cells.

The effect of conjugated linoleic acid on the antioxidant enzyme defense system in rat hepatocytes.

Short-term effects of physiological concentrations of conjugated linoleic acid (CLA) on membrane integrity, metabolic function, cellular lipid composition, lipid peroxidation, and antioxidant enzymes were examined using rat hepatocyte suspension cultures. Incubation with CLA (5-20 ppm) for 3 h decreased the ability of hepatocyte plasma membranes to exclude trypan blue by approximately 25%, and caused leakage of cytosolic lactate dehydrogenase (LDH) into the medium. The significant decrease (P< 0.02) in hepatocyte viability as measured by LDH leakage during cell incubation with 10 and 20 ppm CLA was not associated with significant changes in cellular ATP content. Protein synthesis in hepatocytes was elevated (P < 0.05) in the presence of 5 and 10 ppm CLA, but at a higher concentration (20 ppm), protein synthesis was similar to that of control cells. Gluconeogenesis was maintained in cells incubated with lower concentrations of CLA (5 and 10 ppm) but was decreased (P < 0.02) at the higher concentration. Incubation with 20 ppm CLA for 3 h did not affect the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme of cholesterol synthesis. Both cis-9,trans-11/trans-9,cis-11, and cis-10,trans-12/trans-10,cis-12 isomers of CLA were incorporated to a similar level into hepatocytes. Levels ranged from 3.9 to 4.1%, respectively, of total fatty acids in neutral lipids, and from 0.7 to 0.8%, respectively, of total fatty acids in phospholipids. Cellular lipid peroxidation remained unchanged in the presence of CLA (5-20 ppm), despite significant inhibition (P < 0.05) of superoxide dismutase. Catalase activity was maintained near control levels in the presence of 5 and 10 ppm CLA but was significantly decreased in the presence of 20 ppm CLA. Glutathione peroxidase activity was significantly decreased in the presence of 10 ppm CLA. The apparent sensitivity of the antioxidant enzyme defense system of liver cells to CLA, coupled with the lack of effect of CLA on lipid peroxidation in cells, suggests that cytotoxic effects of CLA as described by LDH leakage and decreased gluconeogenesis were not mediated by a prooxidant action in hepatocytes.

Antioxidant enzyme defence responses of human MCF-7 and SW480 cancer cells to conjugated linoleic acid.

The relationship between antioxidant-enzyme defence responses and cellular growth suppression in human MCF-7 (breast) and SW480 (colon) cancer cells, exposed to CLA in culture was studied. MCF-7 and SW480 cells (1 x 10(6)/flask) were cultured in appropriate medium for 4, 8 and 12 days with varying levels of CLA (0-30 ppm). A dose-dependent decrease in cell numbers and increase in lipid peroxidation, as determined by thiobarbituric acid reactive substances (TBARS) was observed in both cell lines following incubation with CLA. Exposure of both cell lines to 20 ppm CLA for 2-6 days produced a reduction (83-91%) in 3H-leucine incorporation into protein while 3H-uridine and 3H-thymidine incorporation into RNA and DNA were reduced by 49-91% and 86-98%, respectively, compared with untreated control cells. The activities of superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were induced in both cell lines exposed to CLA (20 ppm) over a period of 12 days, although to a greater extent in MCF-7 cells than in SW480 cells. The data indicate that CLA-induced cytotoxicity against MCF-7 and SW480 cancer cell lines is related to the extent of lipid peroxidation of CLA treated cells and affirm that the CLA- induced antioxidant enzymes failed to protect these cells from cytotoxic lipid peroxidation products.

Alpha-tocopherol inhibits oxidative stress induced by cholestanetriol and 25-hydroxycholesterol in porcine ovarian granulosa cells.

The cytotoxicity of oxysterols including 7-ketocholesterol, alpha-epoxide, cholestanetriol and 25-hydroxycholesterol and the possible protecting effect of alpha-tocopherol on cholestanetriol and 25-hydroxycholesterol-induced cytotoxicity were investigated in primary cultures of porcine ovarian granulosa cells. Cell viability as determined by % trypan blue staining and mitochondrial function as determined using 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) reduction were decreased significantly after 24 h exposure to 2.5 microM alpha-epoxide, cholestanetriol and 25-hydroxycholesterol. 7-Ketocholesterol (2.5 microM) did not affect cell viability or mitochondrial function under the same culture conditions. The specific activities of catalase and superoxide dismutase, two antioxidant defense enzymes were increased significantly (p < 0.01) following 24 h exposure to 2.5 microM concentrations of cholestanetriol while only superoxide dismutase was increased in 25-hydroxycholesterol-treated cells (p < 0.001). Specific activity of glutathione peroxidase was unchanged relative to control cells. Levels of thiobarbituric acid reactive substances remained unchanged after exposure to 7-ketocholesterol, alpha-epoxide, cholestanetriol, 25-hydroxycholesterol and cholesterol. Administration of 1 microM alpha-tocopherol to the culture medium significantly improved cell viability and restored both superoxide dismutase and catalase activities to control levels in cholestanetriol -treated cells and only superoxide dismutase in 25-hydroxycholesterol-treated cells. These studies suggest that the cytotoxic nature of physiologically relevant concentrations of cholestanetriol and 25-hydroxycholesterol in granulosa cells is in part due to oxidative stress, but it may be reduced in the presence of alpha-tocopherol.

The response of the antioxidant defense system in rat hepatocytes challenged with oxysterols is modified by Covi-ox.

Suspension cultures of isolated rat hepatocytes were used to investigate whether 7-ketocholesterol and cholestane-3beta,5alpha,6beta-triol exert oxidative stress in cells as manifested by increased lipid peroxidation and the induction of the antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase. The oxysterols were found to increase the levels of both superoxide dismutase and catalase and to have variable effects on glutathione peroxidase activity. Increased lipid peroxidation was not observed, indicating that the endogenous antioxidant defense system was capable of protecting against any oxidative stress that might otherwise by exerted by 7-ketocholesterol or cholestane-3beta,5alpha,6beta-triol. Covi-ox, a natural tocopherol blend reduced the effects of both oxysterols on the antioxidant enzymes. A concurrent reduction in the production of thiobarbituric acid-reactive substances in Covi-ox-treated cells is indirect evidence that reactive oxygen species were produced by oxysterols in hepatocyte suspension cultures.

Elevation of conjugated cis-9, trans-11-octadecadienoic acid in bovine milk because of dietary supplementation.

Cows on pasture were fed full fat soybeans (toasted, flaked, and pelleted) or ground full fat rapeseeds to investigate effects on cis-9, trans-11-octadecadienoic acid in milk. Three herds of 16 cows each that were on pasture were fed 3.1 kg/d of unmolassed beet pulp (control), 3.0 kg/d of rapeseed concentrate, or 3.1 kg/d of a soybean supplement. The concentration of cis-9, trans-11-octadecadienoic acid in the milk of cows fed the rapeseed and soybean supplements was significantly higher than in the milk of cows fed the control diet during the feeding trial. Over the trial, the cis-9, trans-11-octadecadienoic acid concentration in the milk of individual cows varied from 6.8 to 25.7 mg/g of fat in the control herd, from 10.6 to 33.5 mg/g of fat in the herd fed the rapeseed concentrate, and from 8.8 to 30.5 mg/g of fat in the herd fed the soybean supplement. The concentration of cis-9, cis-12-octadecadienoic acid, the substrate for cis-9, trans-11-octadecadienoic acid synthesis in the rumen, was 4.9 g/100 g of fatty acid methyl esters in the milk fat of cows fed the soybean supplement, 2.5 g/100 g of fatty acid methyl esters in the milk fat of cows fed the rapeseed concentrate, and 2.3 g/100 g of fatty acid methyl esters in the milk fat of the control cows. Milk yield and milk constituent yields were not affected by supplementation of either full fat soybeans or rapeseeds compared with controls, but milk protein concentration was significantly reduced by both oilseed supplements.

A direct two-site peroxidase-based enzyme-linked immunosorbent assay for proinsulin.

A non-competitive sandwich assay using an anti-C-peptide IgG and an anti-insulin IgG was developed to measure fasting levels of proinsulin in human serum. The former antibody provided the lower layer in a sandwich immunoassay, the upper layer being composed of an anti-insulin IgG-horse radish peroxidase conjugate. The assay showed negligible cross reactivity at supraphysiological levels of insulin and C-peptide. The method enabled the estimation of proinsulin in fasting non-diabetic control subjects [13.7 +/- 1.6(4) pM] and in type 2 non-insulin-dependent diabetic patients [23.2 +/- 1.1(8) pM].

Alterations in cellular cholesterol metabolism following administration of 6-hydroxydopamine to rabbits.

1. The role of adrenergic mechanisms in the regulation of cholesterol metabolism was investigated by studying the effects of 6-hydroxydopamine (6-OHDA) on serum cholesterol levels and on the activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase, acyl coenzyme A: cholesterol-O-acyl-transferase (ACAT) in the livers and intestines, and cholesterol 7 alpha-hydroxylase in the livers of male New Zealand White rabbits. 2. Total serum cholesterol levels were significantly reduced (P less than 0.01) in 6-OHDA-treated animals. This was reflected in the very low density lipoprotein, low density lipoprotein and high density lipoprotein fractions. The reduction in lipoprotein cholesterol levels reflected reduced cholesterol proportions in the lipoprotein fractions. 3. The 6-OHDA-treated animals also had significantly lower activities of intestinal (P less than 0.001) and hepatic (P less than 0.01) HMGCoA reductase. The specific activities of intestinal ACAT, hepatic ACAT and cholesterol 7 alpha-hydroxylase were comparable in both groups. 4. In contrast to the results observed in vivo, 6-OHDA did not have any in vitro effect on cholesterol biosynthesis in cultured human leucocytes. 5. This latter finding suggests that the effects of 6-OHDA on cellular cholesterol biosynthesis in vivo are indirect, possibly resulting from the known toxic effect of this drug in sympathetic nerve terminals, and imply a potential role for the sympathetic nervous system in the regulation of cellular cholesterol biosynthesis in vivo.