The experience with colorectal cancer screening in the Czech Republic: the detection at earlier stages and improved clinical outcomes.

OBJECTIVES: Colorectal cancer (CRC) remains a major health burden. Although screening is recommended and considered beneficial, further data on its positive effects are needed for worldwide implementation. STUDY DESIGN: The aim of our national multicentre prospective observational study was to reveal and document clinicopathological differences in CRC diagnosed by screening and presented by disease symptoms as well as assess the efficiency of the screening programme in the Czech Republic. METHODS: Between March 2013 and September 2015, a total of 265 patients were enrolled in 12 gastroenterology centres across the Czech Republic. Patients were divided into screening and symptomatic groups and compared for pathology status and clinical characteristics. Screening was defined as a primary screening colonoscopy or a colonoscopy after a positive faecal occult blood test in an average-risk population. RESULTS: The distribution of CRC stages was significantly (statistically and clinically) favourable in the screening group (predominance of stages 0, I and II) compared with the non-screening group (P < 0.001). The presence of distant and local metastases was significantly less frequent in the screening group than in the symptomatic group (P < 0.001). Patients in the screening group had a higher probability of radical surgery (R0) than those diagnosed based on symptoms (P < 0.001). Systemic palliative treatment was indicated in two patients in the screening group compared with 23 patients in the non-screening group (P = 0.018). CONCLUSION: CRC diagnosed by screening disclosed less advanced clinicopathological characteristics and results in patients with a higher probability of radical surgery (R0) than diagnoses established based on symptoms, with subsequent management differing accordingly between both groups. These results advocate the implementation of a suitable worldwide screening programme.

Short communication: Milk fat depression induced by conjugated linoleic acid and a high-oil and low-fiber diet occurs equally across the day in Holstein cows.

Recently, a circadian rhythm of milk and milk component synthesis has been characterized that is partially dependent on the timing of feed intake. Our objective was to determine if inhibition of milk fat synthesis during diet-induced milk fat depression occurred to a higher degree during certain phases of the day. A retrospective analysis was conducted on 2 experiments that induced milk fat depression while milking cows 3 times per day at equal intervals. The response at each milking was analyzed using mixed model ANOVA with repeated measures. In experiment 1, nine multiparous Holstein cows were arranged in a 3×3 Latin square design, and treatments were control, 3-d intravenous infusion of 10 g/d of trans-10,cis-12 conjugated linoleic acid (CLA), and a low-forage and high-fat diet for 10 d. In experiment 2, ten multiparous ruminally cannulated cows were arranged in a replicated design and milk samples were collected during a control period or after 5 d of abomasal infusion of 10 g/d of CLA. The daily pattern of milk fat concentration and yield did not differ between treatments in either experiment. In experiment 1, an effect was found of treatment and milking time on milk fat concentration and yield. Similarly, in experiment 2, main effects were found of treatment and milking time on milk fat concentration and an effect of treatment, but no effect of milking time on milk fat yield. Milk fat percent was increased from 3.41 to 4.06% and 3.25 to 3.48% from the morning to the afternoon milking in experiments 1 and 2, respectively. Additionally, milk fatty acid profile, including trans intermediates, was changed over the day in experiment 1, but the magnitude of the changes were small and the pattern did not differ among treatments. A daily rhythm of milk fat concentration and yield was observed in cows milked 3 times a day, but milk fat depression decreases milk fat yield equally over the day.

Short communication: effect of trans-10,cis-12 conjugated linoleic acid on activation of lipogenic transcription factors in bovine mammary epithelial cells.

The objective of this study was to examine the effect of trans-10,cis-12 conjugated linoleic acid (t10c12CLA) on the activation of transcription factors that potentially regulate lipid synthesis in a bovine mammary epithelial cell line (MAC-T). Cells were transfected with luciferase reporter constructs containing sterol response element (SRE and SRE complex) for sterol regulatory element binding protein-1, peroxisome proliferator response element for peroxisome proliferator-activated receptor γ, or liver X receptor response element for liver X receptor. Different concentrations of t10c12CLA (0, 25, 50, 75, or 100µM) were applied to cells to determine the activation of transcription factors. The influence of t10c12CLA bond structure on transcription factor activation was also investigated by treating cells with different 18:1 fatty acid isomers (trans-10 18:1 or cis-12 18:1) at 100µM. Cells were harvested for luciferase assay after 24h of treatment. Compared with linoleic acid and cis-9,trans-11 CLA controls, the SRE reporters had significantly lower activity in t10c12CLA-treated cells at 50 and 75µM for SRE complex and SRE, respectively. Lower SRE and SRE complex activation was observed in t10c12CLA treatment at 25, 50, and 75µM compared with 0µM. The peroxisome proliferator response element and liver X receptor response element reporters did not respond differently between the t10c12CLA treatment and controls. Compared with t10c12CLA, both trans-10 18:1 and cis-12 18:1 increased the activities of SRE and SRE complex reporters by 1.3- to 4.2-fold. In conclusion, t10c12CLA has an inhibitory role in lipogenic transcription factor activation of SRE, and this negative effect is due to the conjugation of trans-10 and cis-12 double bonds in the fatty acid. Furthermore, we found no support for a regulatory role of response elements for peroxisome proliferator-activated receptor γ or liver X receptor in the t10c12CLA inhibition of mammary lipid synthesis.

Effect of linoleic acid and dietary vitamin E supplementation on sustained conjugated linoleic acid production in milk fat from dairy cows.

Conjugated linoleic acid (CLA; cis-9,trans-11 18:2), a bioactive fatty acid (FA) found in milk and dairy products, has potential human health benefits due to its anticarcinogenic and antiatherogenic properties. Conjugated linoleic acid concentrations in milk fat can be markedly increased by dietary manipulation; however, high levels of CLA are difficult to sustain as rumen biohydrogenation shifts and milk fat depression (MFD) is often induced. Our objective was to feed a typical Northeastern corn-based diet and investigate whether vitamin E and soybean oil supplementation would sustain an enhanced milk fat CLA content while avoiding MFD. Holstein cows (n=48) were assigned to a completely randomized block design with repeated measures for 28 d and received 1 of 4 dietary treatments: (1) control (CON), (2) 10,000 IU of vitamin E/d (VE), (3) 2.5% soybean oil (SO), and (4) 2.5% soybean oil plus 10,000 IU of vitamin E/d (SO-VE). A 2-wk pretreatment control diet served as the covariate. Milk fat percentage was reduced by both high-oil diets (3.53, 3.56, 2.94, and 2.92% for CON, VE, SO, and SO-VE), whereas milk yield increased significantly for the SO-VE diet only, thus partially mitigating MFD by oil feeding. Milk protein percentage was higher for cows fed the SO diet (3.04, 3.05, 3.28, and 3.03% for CON, VE, SO, and SO-VE), implying that nutrient partitioning or ruminal supply of microbial protein was altered in response to the reduction in milk fat. Milk fat concentration of CLA more than doubled in cows fed the diets supplemented with soybean oil, with concurrent increases in trans-10 18:1 and trans-11 18:1 FA. Moreover, milk fat from cows fed the 2 soybean oil diets had 39.1% less de novo synthesized FA and 33.8% more long-chain preformed FA, and vitamin E had no effect on milk fat composition. Overall, dietary supplements of soybean oil caused a reduction in milk fat percentage and a shift in FA composition characteristic of MFD. Supplementing diets with vitamin E did not overcome the oil-induced reduction in milk fat percentage or changes in FA profile, but partially mitigated the reduction in fat yield by increasing milk yield.

t10,c12-CLA decreases adiposity in peripubertal mice without dose-related detrimental effects on mammary development, inflammation status, and metabolism.

The trans 10, cis 12-conjugated linoleic acid (10,12-CLA) isomer reduces adiposity in several animal models. In the mouse, however, this effect is associated with adipose tissue inflammation, hyperinsulinemia and hepatic lipid accumulation. Moreover, 10,12-CLA was recently shown to promote mammary ductal hyperplasia and ErbB2/Her2-driven mammary cancer in the mouse. Reasons for detrimental effects of 10,12-CLA on the mouse mammary gland could relate to its effect on the mammary fat pad (MFP), which is essential for normal development. Accordingly, we hypothesized that mammary effects of 10,12-CLA were mediated through the MFP in a dose-dependent manner. Female FVB mice were fed 10,12-CLA at doses of 0%, 0.1%, 0.2%, or 0.5% of the diet from day 24 of age, and effects on mammary development and metabolism were measured on day 49. The 0.5% dose reduced ductal elongation and caused premature alveolar budding. These effects were associated with increased expression of inflammatory markers and genes shown to alter epithelial growth (IGF binding protein-5) and alveolar budding (TNF-α and receptor of activated NF-κB ligand). The 0.5% dose also caused hyperinsulinemia and hepatic lipid accumulation. In contrast, the 0.1% 10,12-CLA dose had no adverse effects on mammary development, metabolic events, and inflammatory responses, but remained effective in decreasing adipose weights and lipogenic gene expression. These results show that a low dose of 10,12-CLA reduces adiposity in the mouse without negative effects on mammary development, inflammation, and metabolism, and suggest that previously reported detrimental effects relate to the use of excessive doses.

Effects of dietary supplementation of rumen-protected conjugated linoleic acid to grazing cows in early lactation.

Conjugated linoleic acids (CLA) are potent anticarcinogens in animal and in vitro models as well as inhibitors of fatty acid synthesis in mammary gland, liver, and adipose tissue. Our objective was to evaluate long-term CLA supplementation of lactating dairy cows in tropical pasture on milk production and composition and residual effects posttreatment. Thirty crossbred cows grazing stargrass (Cynodon nlemfuensis Vanderyst var. nlemfüensis) were blocked by parity and received 150 g/d of a dietary fat supplement of either Ca-salts of palm oil fatty acids (control) or a mixture of Ca-salts of CLA (CLA treatment). Supplements of fatty acids were mixed with 4 kg/d of concentrate. Grazing plus supplements were estimated to provide 115% of the estimated metabolizable protein requirements from 28 to 84 d in milk (treatment period). The CLA supplement provided 15 g/d of cis-9,trans-11 and 22g of cis-10,trans-12. Residual effects were evaluated from 85 to 112 d in milk (residual period) when cows were fed an 18% crude protein concentrate without added fat. The CLA treatment increased milk production but reduced milk fat concentration from 2.90 to 2.14% and fat production from 437 to 348 g/d. Milk protein concentration increased by 11.5% (2.79 to 3.11%) and production by 19% (422 to 504 g/d) in the cows fed CLA. The CLA treatment decreased milk energy concentration and increased milk volume, resulting in unchanged energy output. Milk production and protein concentration and production were also greater during the residual period for the CLA-treated cows. The CLA treatment reduced production of fatty acids (FA) of all chain lengths, but the larger effect was on short-chain FA, causing a shift toward a greater content of longer chain FA. The CLA treatment increased total milk CLA content by 30% and content of the trans-10,cis-12 CLA isomer by 88%. The CLA treatment tended to decrease the number of days open, suggesting a possible effect on reproduction. Under tropical grazing conditions, in a nutritionally challenging environment, CLA-treated cows decreased milk fat content and secreted the same amount of milk energy by increasing milk volume and milk protein production.

Hot topic: Enhancing omega-3 fatty acids in milk fat of dairy cows by using stearidonic acid-enriched soybean oil from genetically modified soybeans.

Very long chain n-3 fatty acids such as eicosapentaenoic acid (EPA; 20:5n-3) are important in human cardiac health and the prevention of chronic diseases, but food sources are limited. Stearidonic acid (SDA; 18:4n-3) is an n-3 fatty acid that humans are able to convert to EPA. In utilizing SDA-enhanced soybean oil (SBO) derived from genetically modified soybeans, our objectives were to examine the potential to increase the n-3 fatty acid content of milk fat and to determine the efficiency of SDA uptake from the digestive tract and transfer to milk fat. Three multiparous, rumen-fistulated Holstein cows were assigned randomly in a 3 x 3 Latin square design to the following treatments: 1) control (no oil infusion); 2) abomasal infusion of SDA-enhanced SBO (SDA-abo); and 3) ruminal infusion of SDA-enhanced SBO (SDA-rum). The SDA-enhanced SBO contained 27.1% SDA, 10.4% alpha-linolenic acid, and 7.2% gamma-linolenic acid. Oil infusions provided 57 g/d of SDA with equal amounts of oil infused into either the rumen or abomasum at 6-h intervals over a 7-d infusion period. Cow numbers were limited and no treatment differences were detected for DMI or milk production (22.9+/-0.5 kg/d and 32.3+/-0.9 kg/d, respectively; least squares means +/- SE), milk protein percentage and yield (3.24+/-0.04% and 1.03+/-0.02 kg/d), or lactose percentage and yield (4.88+/-0.05% and 1.55+/-0.05 kg/d). Treatment also had no effect on milk fat yield (1.36+/-0.03 kg/d), but milk fat percentage was lower for the SDA-rum treatment (4.04+/-0.04% vs. 4.30+/-0.04% for control and 4.41+/-0.05% for SDA-abo). The SDA-abo treatment increased n-3 fatty acids to 3.9% of total milk fatty acids, a value more than 5-fold greater than that for the control. Expressed as a percentage of total milk fatty acids, values (least squares means +/- SE) for the SDA-abo treatment were 1.55+/-0.03% for alpha-linolenic acid (18:3n-3), 1.86+/-0.02 for SDA, 0.23 +/- <0.01 for eicosatetraenoic acid (20:4n-3), and 0.18+/-0.01 for EPA. Transfer efficiency of SDA to milk fat represented 39.3% (range=36.8 to 41.9%) of the abomasally infused SDA and 47.3% (range=45.0 to 49.6%) when the n-3 fatty acids downstream from SDA were included. In contrast, transfer of ruminally infused SDA to milk fat averaged only 1.7% (range=1.3 to 2.1%), indicating extensive rumen biohydrogenation. Overall, results demonstrate the potential to use SDA-enhanced SBO from genetically modified soybeans combined with proper ruminal protection to achieve impressive increases in the milk fat content of SDA and other n-3 fatty acids that are beneficial for human health.

Effect of supplementation with calcium salts of fish oil on n-3 fatty acids in milk fat.

Enrichment of milk fat with n-3 fatty acids, in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), may be advantageous because of their beneficial effects on human health. In addition, these fatty acids play an important role in reproductive processes in dairy cows. Our objective was to evaluate the protection of EPA and DHA against rumen biohydrogenation provided by Ca salts of fish oil. Four Holstein cows were assigned in a Latin square design to the following treatments: 1) ruminal infusion of Ca salts of fish oil and palm fatty acid distillate low dose (CaFO-1), 2) ruminal infusion of Ca salts of fish oil and palm fatty acid distillate high dose (CaFO-2), 3) ruminal infusion of fish oil high dose (RFO), and 4) abomasal infusion of fish oil high dose (AFO). The high dose of fish oil provided approximately 16 and approximately 21 g/d of EPA and DHA, respectively, whereas the low dose (CaFO-1) provided 50% of these amounts. A 10-d pretreatment period was used as a baseline, followed by 9-d treatment periods with interceding intervals of 10 d. Supplements were infused every 6 h, milk samples were taken the last 3 d, and plasma samples were collected the last day of baseline and treatment periods. Milk fat content of EPA and DHA were 5 to 6 times greater with AFO, but did not differ among other treatments. Milk and milk protein yield were unaffected by treatment, but milk fat yield and DM intake were reduced by 20 and 15%, respectively, by RFO. Overall, results indicate rumen biohydrogenation of long chain n-3 fatty acids was extensive, averaging >85% for EPA and >75% for DHA for the Ca salts and unprotected fish oil supplements. Thus, Ca salts of fish oil offered no protection against the biohydrogenation of EPA and DHA beyond that observed with unprotected fish oil; however, the Ca salts did provide rumen inertness by preventing the negative effects on DM intake and milk fat yield observed with unprotected fish oil.

Evaluation of the mechanism of action of conjugated linoleic acid isomers on reproduction in dairy cows.

The objective of this study was to evaluate the mechanism of action through which conjugated linoleic acid (CLA) beneficially affects reproduction. Lactating Holstein cows (n = 45, 20 +/- 1 DIM) were assigned to 1 of 3 treatments: 70 g/d of Ca salts of tallow (control); 63 g/d of lipid-encapsulated CLA providing 7.1 g/d of cis-9, trans-11 CLA and 2.4 g/d of trans-10, cis-12 CLA (CLA 75:25); or 76 g/d of lipid-encapsulated CLA providing 7.1 g/d each of cis-9, trans-11 and trans-10, cis-12 CLA (CLA 50:50). Supplements were top-dressed for 37 d, milk production and DMI were recorded daily, and blood samples were taken 3 times per week. At 30 +/- 3 DIM, ovulation was synchronized in all cows with a modified Ovsynch protocol, and on d 15 of the cycle cows received an oxytocin injection; blood samples were obtained frequently to measure 13,14 dihydro, 15-keto PGF2alpha. On d 16 of the cycle cows received a PGF2alpha injection and ovarian follicular aspiration was performed 54 h later. Follicular fluid was analyzed for fatty acids, progesterone, and estradiol. Endometrial biopsies were taken before and again near the end of the supplementation period for fatty acid analysis. The CLA resulted in decreased milk fat content of 14.1 and 6.1% at wk 5 of treatment of CLA 50:50 and CLA 75:25, respectively. There were no differences in energy balance or plasma nonesterified fatty acids; however, plasma IGF-I was greater in cows supplemented with CLA 50:50. The CLA isomers were not detectable in endometrial tissue, but cis-9, trans-11 CLA tended to be greater in follicular fluid of supplemented cows. Response to the oxytocin challenge was not different among treatments. Progesterone during the early luteal phase and the estradiol:progesterone ratio in follicular fluid tended to be greater in cows supplemented with CLA 50:50. Overall, these results indicate that short periods of CLA supplementation do not alter uterine secretion of PGF2alpha. The mechanism through which CLA affects reproduction may involve improved ovarian follicular steroidogenesis and increased circulating concentrations of IGF-I.

Effects of feeding fish meal and n-3 fatty acids on ovarian and uterine responses in early lactating dairy cows.

The study was designed to test the effects of dietary supplementation with fish meal or specific n-3 fatty acids on ovarian activity and uterine responses in early lactating cows. From 5 to 50 d in milk (DIM), cows were fed diets that were isonitrogenous, isoenergetic, and isolipidic containing none (control), 1.25, 2.5, or 5% menhaden fish meal (FM) or 2.3% Ca salts of fish oil fatty acids (CaFOFA). Ovarian follicular dynamics were monitored along with plasma concentrations of estradiol and progesterone. Beginning at 23 DIM, cows were induced into a synchronized ovulatory cycle. On d 15 after ovulation (49 DIM), cows were injected with oxytocin and blood samples were collected to monitor uterine release of PGF(2alpha) (measured as 13, 14-dihydro-15-keto PGF(2alpha); PGFM). Uterine endometrial biopsies were collected for fatty acid analysis and cyclooxygenase-2 (COX-2) protein measurement. Ovarian follicular activities as well as plasma estradiol and progesterone concentrations were similar across diets. Endometrial fatty acid composition of eicosapentaenoic acid (C20:5, n-3) and docosahexaenoic acid (C22:6, n-3) were increased as much as 3-fold by supplementation with fish meal and CaFOFA. Conjugated linoleic acid (C18:2 cis-9, trans-11) in the endometrium was also increased; conversely, arachidonic acid (C20:4, n-6) percentage was decreased by 5% FM. Plasma PGFM response to oxytocin injection was not different among diets and endometrial COX-2 protein abundance did not differ. Results from this experiment demonstrate that dietary supplementation with fish meal or n-3 fatty acids in early lactating dairy cows significantly increased uterine n-3 fatty acid concentrations, but had no apparent effect on endometrial COX-2 or PGF(2alpha) production in response to oxytocin challenge.

Comparison of calcium salts and formaldehyde-protected conjugated linoleic Acid in inducing milk fat depression.

Abomasal infusion studies have shown that trans-10, cis-12 conjugated linoleic acid (CLA) decreases milk fat synthesis. However, supplements of CLA must avoid rumen biohydrogenation for this technology to be applied to ruminants. Rumen protection methods would reduce CLA metabolism in the rumen and increase its supply to the small intestine. Our objective was to compare the efficacy of 2 forms of rumen-protected CLA at inducing milk fat depression. Three mid to late lactation Holstein cows each fitted with a rumen fistula were used in a 3 x 3 Latin square design. Treatments were: 1) control, 2) calcium salts of CLA (Ca-CLA), and 3) formaldehyde-protected CLA (FP-CLA). Supplements were designed to provide 10 g/d of trans-10, cis-12 CLA and were administered intraruminally once per day to ensure exact delivery of amount. Both CLA treatments substantially reduced milk fat yield and content compared with control, with the reductions in milk fat yield averaging 34% for the Ca-CLA treatment and 44% for the FP-CLA treatment. In contrast, milk yield, milk protein yield, and dry matter intake were unaltered by CLA treatment. Efficiency of transfer of trans-10, cis-12 CLA from the supplement into milk fat was 3.2 and 7.0% for Ca-CLA and FP-CLA, respectively. These values are much lower than transfer efficiencies reported for abomasally infused CLA, suggesting that much of the trans-10, cis-12 CLA present in the 2 formulations was biohydrogenated in the rumen. Overall, the extent of the reduction in milk fat yield indicates that both protection formulations are acceptable methods for the formulation of CLA supplements to induce milk fat depression in lactating dairy cows.

trans-10, cis-12 conjugated linoleic acid decreases lipogenic rates and expression of genes involved in milk lipid synthesis in dairy cows.

Feeding conjugated linoleic acid (CLA) reduces milk fat synthesis in lactating dairy cows, and the effect has been shown to be specific for the trans-10, cis-12 CLA isomer. Our objectives were to examine potential mechanisms by which trans-10, cis-12 CLA inhibits milk fat synthesis. Multiparous Holstein cows (n = 4) in late lactation were used in a balanced 2 x 2 crossover design. Treatments consisted of a 5 d abomasal infusion of either skim milk (control) or purified trans-10, cis-12 CLA (13.6 g/d) emulsified in skim milk. On d 5 of infusion, mammary gland biopsies were performed and a portion of the tissue analyzed for mRNA expression of acetyl CoA carboxylase, fatty acid synthetase, delta 9-desaturase, lipoprotein lipase, fatty acid binding protein, glycerol phosphate acyltransferase and acylglycerol phosphate acyltransferase. Lipogenic capacity was evaluated with another portion of the tissue. Infusion of trans-10, cis-12 CLA decreased milk fat content and yield 42 and 48%, respectively and increased the trans-10, cis-12 CLA content in milk fat from < 0.1 to 4.9 mg/g. Reductions in milk fat content of C4 to C16 fatty acids contributed 63% to the total decrease in milk fat yield (molar basis). Analysis of the ratios of specific fatty acid pairs indicated trans-10, cis-12 CLA also shifted fatty acid composition in a manner consistent with a reduction in delta 9-desaturase. Mammary explant incubations with radiolabeled acetate established that lipogenic capacity was decreased 82% and acetate oxidation to CO2 was reduced 61% when cows received trans-10, cis-12 CLA. Infusing trans-10, cis-12 CLA also decreased the mRNA expression of all measured enzymes by 39 to 54%. Overall, data demonstrated the mechanism by which trans-10, cis-12 CLA inhibits milk fat synthesis includes decreasing expression of genes that encode for enzyme involved in circulating fatty acid uptake and transport, de novo fatty acid synthesis, desaturation of fatty acids and triglyceride synthesis.

Isomer-specific antidiabetic properties of conjugated linoleic acid. Improved glucose tolerance, skeletal muscle insulin action, and UCP-2 gene expression.

Conjugated linoleic acid (CLA) isomers have a number of beneficial health effects, as shown in biomedical studies with animal models. Previously, we reported that a mixture of CLA isomers improved glucose tolerance in ZDF rats and activated peroxisome proliferator-activated receptor (PPAR)-gamma response elements in vitro. Here, our aim was to elucidate the effect(s) of specific CLA isomers on whole-body glucose tolerance, insulin action in skeletal muscle, and expression of genes important in glucose and lipid metabolism. ZDF rats were fed either a control diet (CON), one of two CLA supplemented diets (1.5% CLA) containing differing isoforms of CLA (47% c9,t11; 47.9% c10,t12, 50:50; or 91% c9,t11, c9,t11 isomers), or were pair-fed CON diet to match the intake of 50:50. The 50:50 diet reduced adiposity and improved glucose tolerance compared with all other ZDF treatments. Insulin-stimulated glucose transport and glycogen synthase activity in skeletal muscle were improved with 50:50 compared with all other treatments. Neither phosphatidlyinositol 3-kinase activity nor Akt activity in muscle was affected by treatment. Uncoupling protein 2 in muscle and adipose tissue was upregulated by c9,t11 and 50:50 compared with ZDF controls. PPAR-gamma mRNA was downregulated in liver of c9,t11 and pair-fed ZDF rats. Thus, the improved glucose tolerance in 50:50 rats is attributable to, at least in part, improved insulin action in muscle, and CLA effects cannot be explained simply by reduced food intake.

Control of rat mammary epithelium proliferation by conjugated linoleic acid.

Past research showed that mammary gland morphogenesis in the pubescent rat was retarded by the feeding of conjugated linoleic acid (CLA). A major objective of the present study was to examine the proliferative activity and the expression of cell cycle regulatory proteins in the developing mammary epithelium of rats fed a mixture of CLA isomers (primarily as free fatty acid c9, t11-CLA and t10,c12-CLA) or a highly enriched natural source of c9,t11-CLA (as triacylglycerol in butterfat). In both experiments, the diets, with or without CLA, were started at weaning and continued for four weeks. The two CLA preparations were equally effective in suppressing bromodeoxyuridine labeling and the expression of cyclin D1 and cyclin A (determined by immunohistochemistry) in the terminal end buds and alveolar clusters of the mammary epithelium while it undergoes extensive ductal branching during pubescence. There was a trend of an increase, although not statistically significant, in the proportion of cells expressing the p16 and p27 cdk inhibitors. A separate experiment was designed to evaluate the effect of c9,t11-CLA (as a free fatty acid of > 90% purity) treatment on the rate of proliferation of the mammary epithelium as the animal matured from weanling to adult. The bromodeoxyuridine labeling data indicated that the mammary epithelium appeared to lose its sensitivity to CLA control of proliferation as it completely filled the fat pad and became quiescent. These observations suggest that the responsiveness of mammary epithelial cells to CLA intervention may be dependent on their proliferative status.

Vaccenic acid feeding increases tissue levels of conjugated linoleic acid and suppresses development of premalignant lesions in rat mammary gland.

The objective of this report was to determine whether vaccenic acid (t11-18:1) is converted efficiently to conjugated linoleic acid (c9,t11-18:2, CLA) in rats via the delta 9-desaturase reaction and, if so, whether vaccenic acid could substitute for CLA as an anticancer agent. In Study 1, rats were fed 1%, 2%, or 3% vaccenic acid in their diet, and tissue levels of CLA and CLA metabolites were determined in liver and mammary gland. In general, concentrations of CLA and CLA metabolites increased proportionately with an increase in vaccenic acid intake, at least up to the 2% dose level. Beyond this dose, there was clearly a plateauing effect. Thus vaccenic acid concentration increased from an undetectable level in the control to 78.5 nmol/mg lipid in the liver of rats fed a 2% vaccenic acid diet. This was accompanied by an increase in CLA from 2.3 to 33.6 nmol/mg lipid. These changes were also mirrored in the mammary gland, where increases in vaccenic acid (from 27.5 to 163.2 nmol/mg lipid) and CLA (from 17.8 to 108.9 nmol/mg lipid) were similarly observed. Vaccenic acid at 2% produced a CLA concentration in the mammary gland that was historically associated with a positive response in tumor inhibition based on our past experience. This provided the basis for selecting 2% vaccenic acid in Study 2, which was designed to evaluate its efficacy in blocking the development of premalignant lesions in the rat mammary gland. In this experiment, formation of histologically identifiable pathology due to intraductal proliferation of terminal end bud cells of mammary epithelium was used as the end point of analysis at 6 wk after carcinogen administration. Treatment with vaccenic acid reduced the total number of these premalignant lesions by approximately 50%. We hypothesize that the anticancer response to vaccenic acid is likely to be mediated by its endogenous conversion to CLA via delta 9-desaturase.

Technical note: production of butter with enhanced conjugated linoleic acid for use in biomedical studies with animal models.

Cancer models utilize massive doses of carcinogen so that investigations of anticarcinogenic effects require equally large doses. Conjugated linoleic acids (CLA), predominately consumed in dairy products, are thought to be anticarcinogenic. Our objective was to naturally produce a CLA-enhanced butter for use in biomedical studies with animal models. To do this, we fed cows a low forage diet supplemented with sun-flower oil. This resulted in increases in content of CLA of milk fat, but the markedly elevated concentrations were transient and declined over a 3-wk period. By collecting milk fat over the first few days on the diet (d 7 to 10) and selecting cows with the greatest CLA concentrations, we were able to produce a butter in which CLA content was enhanced sevenfold over control butter (41 vs. 5 mg/g of fatty acids) and the cis-9, trans-11 isomer predominated (91%). Thus, butter produced by this method can be used to investigate the preventive role of CLA in natural foods with biomedical models of different types of cancer. Furthermore, the butter allows examination of the other beneficial health effects of CLA reported with animal models.

Conjugated linoleic acid is synthesized endogenously in lactating dairy cows by Delta(9)-desaturase.

Conjugated linoleic acid (CLA) is a naturally occurring anticarcinogen found in milk fat and body fat of ruminants. Although CLA is an intermediate in ruminal biohydrogenation of linoleic acid, we hypothesized that its primary source was from endogenous synthesis. This would involve Delta(9)-desaturase and synthesis from trans-11 18:1, another intermediate in ruminal biohydrogenation. Our first experiment supplied lactating cows (n = 3) with trans-11 18:1 by abomasal infusion and examined the potential for endogenous synthesis by measuring changes in milk fat CLA. By d 3, infusion of trans-11 18:1 resulted in a 31% increase in concentration of cis-9, trans-11 CLA in milk fat, demonstrating that an active pathway for endogenous synthesis of CLA exists. Our second experiment examined the quantitative importance of endogenous synthesis of CLA in lactating cows (n = 3) by abomasally infusing a putative stimulator (retinol palmitate) or an inhibitor (sterculic oil) of Delta(9)-desaturase. Infusion of retinol palmitate had no influence on milk fatty acid desaturation, and yield of CLA in milk fat was not altered. However, sterculic oil infusion decreased the concentration of CLA in milk fat by 45%. Consistent with Delta(9)-desaturase inhibition, the sterculic oil treatment also altered the milk fat concentration of other Delta(9)-desaturase products as indicated by the two- to threefold increase in the ratios of 14:0 to 14:1(,) 16:0 to 16:1 and 18:0 to cis-18:1. Using changes in the ratio of 14:0 to 14:1 as an indication of the extent of Delta(9)-desaturase inhibition with the sterculic oil treatment, an estimated 64% of the CLA in milk fat was of endogenous origin. Overall, results demonstrate that endogenous synthesis of CLA from trans-11 18:1 represented the primary source of CLA in milk fat of lactating cows.