Dominique Gruffat

Factors influencing proportion and composition of CLA in beef.

Bovine meat is criticised for the bad nutritional image of its lipids and fatty acids. However, with dairy products, beef is the major source of conjugated linoleic acid (CLA) which could have several human health benefits. The present study compared, from data of five nutritional experiments on bovine animals performed by the laboratory, the impact of factors linked to the animals (breed, age, sex, type of muscle) and to feeding conditions (basal diet, lipid supplements) on the CLA proportion and composition in muscles. Among these factors, linseed supplementation was an efficient way to increase CLA proportion in beef (+22% to +36%) but was highly modulated by the nature of the basal diet, and by intrinsic factors (breed, age/sex, type of muscle) since these ones could modulate CLA proportion in beef from 24% to 47%. Moreover, these factors modified also the proportion of cis,trans-CLA, related to cis,cis- and trans,trans-isomers. Specific biological properties of these latter isomers should be determine to understand the consequences of intramuscular CLA isomer variations for the health of consumers.

Beef conjugated linoleic acid isomers reduce human cancer cell growth even when associated with other beef fatty acids.

Although many data are available concerning anticarcinogenic effects of industrial conjugated linoleic acid (CLA), few studies have reported the antitumour properties of CLA mixtures originating from ruminant products. The aim of the present study was to investigate the in vitro antiproliferative effects of beef CLA mixtures on breast, lung, colon, melanoma and ovarian human cancer cell lines. For this purpose, four fatty acid (FA) extracts prepared from beef lipid and varying in their CLA composition, their corresponding purified CLA-enriched fractions, and mixtures of pure synthetic CLA, the composition of which reproduced that of the four selected beef samples, were tested on cancer cell lines. Cancer cells were exposed for 48 h to medium containing 100 microm-FA and their proliferation was determined by quantifying cellular DNA content (Hoechst 33342 dye). Compared with cells incubated without FA, the number of cancer cells was reduced from 25 to 67 % (P<0.0001) following FA treatment. Antiproliferative effects of CLA mixtures varied in magnitude according to the source of FA, the CLA composition and the cell lines. CLA mixtures naturally present in beef inhibited the proliferation of human cancer cell lines, a high content in cis-trans isomers allowing the most important antiproliferative effect. Beef total FA exhibited a greater growth-inhibitory activity than their corresponding CLA-enriched fractions. These results suggested that either beef FA other than beef CLA could possess antiproliferative properties and/or the existence of complementary effects of non-conjugated FA and CLA, which could favour the antiproliferative properties of beef total FA.

Plant extracts combined with vitamin E in PUFA-rich diets of cull cows protect processed beef against lipid oxidation.

The effect of supplementing PUFA-rich cull cow diets with vitamin E (2.8 g/animal/day) or vitamin E plus plant extracts rich in polyphenols (PERP) (126 g/animal/day), for 101+/-3 days preceding slaughter, on the oxidative stability of longissimus thoracis (LT) and semitendinosus (ST) steaks was evaluated after ageing (for 12 d at 4 degrees C either in carcass or under-vacuum) and packaging (14 d under-vacuum (V), 4 d aerobic (A) and 7 d under modified atmosphere (70:30, O(2)/CO(2)) (MA)). The ageing method had no effect on a beef lipid oxidation intensity marker (malondialdehyde (MDA)), whereas packaging systems containing O(2) (A and MA) significantly increased lipid oxidation intensity (5 and 13 times higher than under V, respectively). Adding antioxidants to diets of animals given a PUFA-rich diet significantly improved lipid stability in steaks; the combination of vitamin E and PERP was more efficient than vitamin E alone for the most deleterious beef packaging.

Lipid absorption and hepatic metabolism in ruminants

Les matikres grasses sont ajoutCes aux rations des ruminants pour ameliorer l’efficacite d’utilisation de 1’Cnergie B des fins de production. L’intestin gr&le des ruminants posskde une forte capacitC d’absorption des acides gras alimentaires saturCs par l’action des hydrogenases bactkriennes du rumen. La synthkse et la secretion des lipoproteines riches en triacylglycerols (chylomicrons, VLDL) par l’intestin grCle varie avec le degri d’insaturation des chaines grasses alimentaires. Le lent processus d’absorption des acides gras chez le veau prkruminant favorise la sCcrCtion des lipoprotkines riches en triacylglycerols par la voie de la veine porte. La fraction protkique de ces particules lipoprot6iques est dominCe, chez le bovin, par l’apoprotkine B48, forme tronquee de l’apo B resultant de la modification unique post-transcriptionnelle de I’ARN messager de l’apo B. Le metabolisme hepatique des lipides des ruminants prCsente des particularites favorisant la formation de foie gras et le dCveloppement d’une cCtoacidose nuisible B la sante et aux capacites de reproduction des animaux. Le foie possede une forte capacite d’estkrification et de stockage des acides gras exogknes sous forme de triacylglycerols ou d’oxydation de ces acides gras sous forme de corps cktoniques. Les faibles capacitCs de synthkse de l’apo BlOO et de mobilisation des triacylglycCrols stock& dans le cytoplasme limitent fortement la secrktion hkpatique des triacylglycerols sous forme de VLDL. Chez les femelles en debut de lactation, le diveloppement de l’infiltration lipidique du foie n’est pas rCduit par la supplementation lipidique des rations mais plut8t par l’apport alimentaire d’acides amines limitants (mkthionine, lysine). La modulation de I’expression du gkne hkpatique de l’apo B au cours du cycle gestation-lactation explique, en partie, le developpement de l’infiltration graisseuse observee en debut de lactation.

Effects of fish oil and additional starch on tissue fatty acid profile and lipogenic gene mRNA abundance in lactating goats fed a diet containing sunflower-seed oil

In dairy cattle, diet supplementation with oils affects the lipid metabolism in body tissues via changes in the partitioning and deposition of fatty acids (FAs) and lipogenic gene expression; however, limited data are available in goats. Eight Alpine goats were fed a grassland hay diet supplemented with 90 g/day of sunflower-seed oil or 90 g/day of sunflower-seed oil and fish oil (2 : 1) plus additional starch. The goats were slaughtered on day 21 of the treatments and samples of the mammary secretory tissue, liver, omental and perirenal adipose tissues (ATs) were collected to characterise their FA composition and the mRNA abundance of lipogenic genes and transcription factors involved in their regulation, and to examine the impact of the diet composition on the same parameters. The results are in agreement with the specific physiological adaptation in the lipid metabolism of body tissues that is likely to occur during late lactation because of the coexistence of an active lipogenesis in the mammary secretory tissue and a significant anabolic activity in the ATs. These latter tissues were characterised by high concentrations of saturated FA and very low polyunsaturated FA (PUFA) levels. The content of PUFA was relatively higher in the mammary secretory tissue, in particular in the case of polyunsaturated C18. The highest PUFA contents were found in the liver, in accordance with the greater mRNA abundances of the genes that encode the necessary enzymes for very long-chain n-3 and n-6 PUFA synthesis. However, substantial differences between n-3 and n-6 pathways would most likely exist in the goat liver. Overall, differences in diet composition induced limited changes in the mRNA abundance of genes involved in lipid metabolism, and these were not associated with the few variations observed in tissue FA composition.

Vaccenic acid metabolism in the liver of rat and bovine.

Hepatic metabolism of vaccenic acid (VA), especially its conversion into CLA, was studied in the bovine (ruminant species that synthesizes CLA) and in the rat (model for nonruminant) by using the in vitro technique of liver explants. Liver tissue samples were collected from fed animals (5 male Wistar rats and 5 Charolais steers) and incubated at 37°C for 17 h under an atmosphere of 95% O2/5% CO2 in medium supplemented with 0.75 mM of FA mixture and with 55 μM [1-14C]VA. VA uptake was about sixfold lower in bovine than in rat liver slices (P<0.01). For both species, VA that was oxidized to partial oxidation products represented about 20% of VA incorporated by cells. The chemical structure of VA was not modified in bovine liver cells, whereas in rat liver cells, 3.2% of VA was converted into 16∶0 and only 0.33% into CLA. The extent of esterification of VA was similar for both animal species (70–80% of incorporated VA). Secretion of VA as part of VLDL particles was very low and similar in rat and bovine liver (around 0.07% of incorporated VA). In conclusion, characteristics of the hepatic metabolism of VA were similar for rat and bovine animals, the liver not being involved in tissue VA conversion into CLA in spite of its high capacity for FA desaturation especially in the rat. This indicates that endogenous synthesis of CLA should take place exclusively in peripheral tissues.

Thyrotropin chronically regulates the pool of thyroperoxidase and its intracellular distribution: A quantitative confocal microscopic study

The regulation of thyroperoxidase (TPO) expression and of its intracellular distribution was studied in porcine thyroid cells cultured on porous bottom filters. Cells were cultured for 18 days in the absence or in the presence of thyrotropin (TSH) and with or without iodide. Microsomes were purified and analyzed by electrophoresis. TPO was detected by immunoblotting with polyclonal anti‐porcine TPO antibodies and quantified by scanning the bands. The amount of TPO was increased 2‐fold by TSH. High concentrations of iodide (1–50 μM, added daily) decreased the level of TPO. Confocal microscopy served to determine the intracellular localization of TPO and its quantitative distribution. Intracellular and surface‐located TPO was detected by fluorescein‐labeled antibodies on saponin‐treated cells. Quantitative confocal microscopy showed that TSH increased the total amount of TPO 2‐fold as for immunoblotting. The highest amount of TPO was found in the perinuclear area and between the nucleus and the Golgi apparatus. Only 4% of TPO was present on the apical surface and about 1% on the basolateral membrane; the remainder (about 95%) was inside the cells. TSH did not change these relative contents. TSH modified the intracellular distribution of the enzyme, increasing the TPO pool from the perinuclear area to apical membrane. This domain could be a site of storage of TPO. Adding a physiological concentration of iodide (0.5 μM, daily) did not influence the intracellular distribution of TPO. We concluded that chronic TSH stimulation (1) increased 2‐fold the pool of TPO but did not change the relative proportion of TPO inside the cells and on the apical surface, and (2) modified the intracellular distribution of vesicular TPO, the major part of which was accumulated in the perinuclear and cytoplasmic area under the subapical domain of the polarized cells. J. Cell. Physiol. 174:160–169, 1998.

Effect of dietary n−6 and n−3 polyunsaturated fatty acids on peroxidizability of lipoproteins in steers

The susceptibility of major plasma lipoproteins to lipoperoxidation was studied in relation to the FA composition of their neutral and polar lipids in steers given PUFA-rich diets. Two trials used, respectively, 18 (“sunflower” experiment, S) or 24 (“linseed” experiment, L) crossbred Salers x Charolais steers. Each involved three dietary treatments over a 70-d period: a control diet (CS or CL diets) consisting of hay and concentrate, or the same diet supplemented with oilseeds (4% diet dry matter) fed either as seeds (SS or LS diets) or continuously infused into the duodenum (ISO or ILO diets). Compared with control diets, ISO and ILO treatments tended to decrease the resistance time of LDL and HDL classes to peroxidation, mainly owing to the enrichment of their polar and neutral lipids with PUFA. With diets SS and LS, sensitivity of major lipoprotein classes (LDL, light and heavy HDL) was not affected because ruminal hydrogenation of dietary PUFA decreased their incorporation into lipoparticles. ISO and ILO treatments induced a more important production of conjugated dienes and hydroperoxides generated by peroxidation in the three lipoprotein classes due to the higher amounts of PUFA esterified in lipids of the core and the hydrophilic envelope of particles. The production of malondialdehyde (MDA) increased in steers fed linseed supplements, indicating that MDA production did not occur with linoleic acid provided by sunflower oil supplements. Thus, plasma peroxidation of PUFA generates toxic products in steers fed diets supplemented with PUFA and can be deleterious for the health of the animal during long-term treatment.

Hepatic metabolism of glucose and linoleic acid varies in relation to susceptibility to fatty liver in ad libitum-fed Muscovy and Pekin ducks

The susceptibility to develop hepatic steatosis is known to differ between duck species, especially between Muscovy and Pekin ducks. This difference could be explained by either differential responses of species to overfeeding or genetic differences in hepatic lipid metabolism. The aim of the present study was to compare the intensities of the different hepatic pathways (oxidation, lipogenesis, esterification, secretion, etc.) of the two main nutrients (glucose and linoleic acid (LA)) reaching the liver of ad libitum-fed Muscovy (n 6) and Pekin (n 6) ducks using the ex vivo method of liver slices incubated for 16 h with [U-14C]glucose, [1-14C]LA and [35S]methionine added to the survival medium. In such experimental conditions, the lipogenesis pathway from glucose was 2-fold higher (P<0.05) in the liver of the Muscovy duck than in that of the Pekin duck. Furthermore, the hepatic uptake of LA was 2-fold higher (P<0.05) in the Muscovy duck than in the Pekin duck leading to a 2-fold higher (P<0.05) esterification of this fatty acid in the liver of the Muscovy duck. The hepatic secretion of VLDL was higher (P<0.01) in the Muscovy duck than in the Pekin duck but insufficient to prevent lipid accumulation in the liver of the Muscovy duck. In conclusion, these results show the influence of the species on the hepatic metabolism of ducks in relation to their susceptibility to develop fatty liver. These results should shed light on the metabolic regulations that might underlie susceptibility to hepatic steatosis in the the human liver.

In vitro comparison of hepatic metaboliosm of 9cis-11 trans and 10trans-12cis isomers of CLA in the rat

Hepatic metabolism of the two main isomers of CLA (9cis-11 trans, 10trans-12cisC18∶2) was compared to that of oleic acid (representative of the main plasma FA) in 16 rats by using the in vitro method of incubated liver slices. Liver tissue samples were incubated at 37°C for 17h under an atmosphere of 95% O2/5%CO2 in a medium supplemented with 0.75 mM of FA mixture (representative of circulating nonesterified FA) and with 55 μM [1-14C]9cis-11 trans C18∶2, [1-14C]10trans-12cis C18∶2, or [1-14C]oleate. The uptake of CLA by hepatocytes was similar for both isomers (9%) and was three times higher (P<0.01) than for oleate (2.6%). The rate of CLA isomer oxidation was two times higher (49 and 40% of incorporated amounts of 9cis-11 trans and 10trans-12 cis, respectively) than that of oleate (P<0.01). Total oxidation of oleate and CLA isomers into [14CO2] was low (2 to 7% of total oxidized FA) compared to the partial oxidation (93 to 98%) leading to the production of [14C] acid-soluble products. CLA isoemrs escaping from catabolism were both highly desaturated (26.7 and 26.8%) into conjugated 18∶3. Oleate and CLA isomers were mainly esterified into neutral lipids (30%). They were slowly secreted as parts of VLDL particles (<0.4% of FA incorporated into cells), the extent of secretion of oleate and of 10trans-12 cis being 2.2-fold higher than that of 9cis-11 trans (P<0.02). In conclusion, this study clearly showed that both CLA isomers were highly catabolized by hepatocytes, reducing their availability for peripheral tissues. Moreover, more than 25% of CLA escaping from catabolism was converted into conjugated 18∶3, the biological properties of which remain to be elucidated.