Fatty acid composition and oxidation in beef muscles as affected by ageing times and cooking methods.

This study aimed to determine how ageing and cooking, each one applied to the beef meat most suitable (pan-fried or grilled ribeye steak, braised chuck and fried or roasted rump steak), induce changes in lipid content, fatty acid (FA) composition and lipid oxidation of muscles from 16 cattle representative of animals raised for France meat production. The fattiest muscle (ribeye) was the richest in saturated and monounsaturated FA leading to poor nutritional indexes. In contrast, the leanest muscle (rump) had the highest proportion of polyunsaturated FA and the highest levels of peroxidation without exceeding critical limits. The impact of cooking methods seemed mainly linked to the moisture loss increasing meat fat content and the culinary fat addition whose FA composition marked the meat. Cooking methods induced oxidation phenomena that could exceed the limit thresholds. In conclusion, short cooking time of rump steak was the best combination to meet nutritional expectations.

Extruded linseed and rapeseed both influenced fatty acid composition of total lipids and their polar and neutral fractions in longissimus thoracis and semitendinosus muscles of finishing Normand cows.

The effects of extruded linseed and rapeseed on lipids and FA composition of total, polar and neutral lipids of longissimus thoracis (LT) and semitendinosus (ST) muscles were investigated in 21 Normand cull cows. Animals were assigned in a 100 d finishing period to straw (30%) and concentrate (70%) based (C) or the same diet supplemented with linseed (L) or with rapeseed (66%) plus linseed (33%) (RL). Beef polar and neutral lipids were purified by liquid chromatography and their FA analysed by GLC. Trans and cis 18:1, purified by HPLC from total FA methyl esters, were analysed by GLC-MS. L and LR diets did not increase beef lipid deposition, but had modified FA composition of both LT and ST muscles in favouring deposition of 18:3n-3 and 9cis,11tr 18:2 (CLA), mainly to the detriment of 18:1∆9 cis (neutral lipids) and 18:2n-6 (polar lipids). However, they did not favour deposition of LC n-3 PUFA in the two muscles, but had increased deposition of trans 18:1 significantly, especially of ∆13tr to ∆16tr isoforms to the detriment of ∆10tr 18:1 (L diet) and of ∆11tr 18:1 (RL diet).

Structural and biochemical characteristics of bovine intramuscular connective tissue and beef quality.

The aim of the study was to evaluate the impact of structural and biochemical characteristics of muscle intramuscular connective tissue on beef quality. The experimental design was based on three muscles of three breeds sampled as fresh material and cooked at 55°C (Longissimus thoracis and Semimembranosus) or at 70°C (Semimembranosus and Biceps femoris) for quality assessment. The results showed that muscle characteristics influence beef quality differently from one muscle to another. In grilled LT, proteoglycan content contributed negatively to juiciness, and intramuscular lipids were linked positively to tenderness, flavour, residues and overall liking scores. In grilled SM, cross-link and lipid contents were involved in beef quality. In BF cooked to 70°C, perimysial branch points were negatively linked to tenderness. In SM cooked to 70°C, perimysial area was involved in beef quality. These results should allow a better understanding of the factors involved in background toughness, in juiciness and flavour of meat.

Relationships between muscle growth potential, intramuscular fat content and different indicators of muscle fibre types in young Charolais bulls.

Genetic selection in favor of muscle growth at the expense of fat should affect characteristics of muscles, and therefore beef quality. This study was conducted with two extreme groups of six animals selected among 64 Charolais young bulls ranked according to their genetic potential for muscle growth. Muscle characteristics were assessed in Rectus abdominis (RA, slow oxidative) and Semitendinosus (ST, fast glycolytic) muscles. Intramuscular fat content and proportions of myosin heavy chains I (slow) and IIA (fast oxido-glycolytic) and certain indicators of oxidative metabolism (activities of citrate synthase (CS), isocitrate dehydrogenase and cytochrome-c oxidase (COX); expression of H-fatty acid binding protein (FABP)) were higher in RA than in ST muscle. Genetic selection for muscle growth reduced intramuscular fat content and the activities of some oxidative metabolism indicators (namely CS, COX only). The positive correlation between muscle triacylglycerol content and A-FABP messenger RNA level (a marker of adipocyte differentiation) (r = 0.53, P < 0.05) suggests that A-FABP may be a good marker of the ability of bovines to deposit intramuscular fat. In conclusion, the metabolic muscle characteristics which respond to the selection process in favor of muscle growth clearly differ from the muscle characteristics which allow muscle types to be differentiated.

Expression of enzymes and transcription factors involved in n-3 long chain PUFA biosynthesis in limousin bull tissues.

The current low consumption of n-3 long chain polyunsaturated fatty acids (n-3 LCPUFA) led scientists to wonder about the possible enrichment of human food, including meats such as beef, with n-3 LCPUFA. However, their biosynthesis from dietary n-3 PUFA seems limited in mammalian tissues implying that a better understanding of the molecular mechanisms responsible for this down regulation is needed. This study aimed at identifying and comparing the limiting steps of n-3 LCPUFA synthesis in liver, intermuscular adipose tissue (IM-AT) and semitendinosus muscle (ST) from six Limousin bulls. Tissue FA composition was analysed by GLC and mRNA abundance of enzymes and transcription factors involved in n-3 LCPUFA synthesis was assessed by RT-qPCR. In liver, mRNA encoding proteins involved in n-3 LCPUFA synthesis were present in agreement with the significant high content of n-3 LCPUFA (8.4 mol% of total FA, 257 mg/100 g of fresh tissue) in this organ. In IM-AT, these mRNA were all present, but at a tenfold lower intensity than in liver in agreement with the low contents of n-3 LCPUFA in this tissue. In ST muscle, these mRNA were all present except elongase 5 mRNA which was only present as trace, the corresponding protein being undetectable, probably inducing a break of n-3 LCPUFA synthesis from 18:4n-3. In conclusion, Limousin bull ST muscle seemed unable to synthesize n-3 LCPUFA. However, the presence of 20:5n-3 (EPA) and 22:5n-3 (DPAn-3) in muscle raised the question of the origin of these n-3 LCPUFA.

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.

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.

Is the hepatic metabolism of glucose and linoleic acid influenced by species in overfed ducks?

There are genetic differences in the hepatic glucose and linoleic acid metabolisms between Muscovy and Pekin ducks ad libitum-fed. To understand the effect of overfeeding on the hepatic metabolisms in these two species of ducks, we compared the different pathways of glucose and linoleic acid reaching the liver of Muscovy (Cairina moschata) (n=6) and Pekin (Anas platyrhynchos) (n=6) ducks overfed for 1 week and sacrificed 2-4 h after their last meal by using the ex vivo method of liver slices incubated for 16 h with [U-(14)C]-glucose, [1-(14)C]-linoleic acid and [(35)S]-methionine added to the survival medium. The glucose was the main precursor of triacylglycerol synthesis in the liver of these two species and its hepatic metabolism was similar between species. The hepatic uptake of linoleic acid was 1.7-fold higher (P=0.020) in the Muscovy duck than in the Pekin duck leading to a 1.9-fold higher (P=0.017) esterification of this fatty acid in the liver of the Muscovy duck than in that of the Pekin duck. Finally, both species after 1 week of overfeeding exhibited the same capacity to secrete VLDL remaining insufficient to avoid hepatic steatosis.

Butters varying in trans 18:1 and cis-9,trans-11 conjugated linoleic acid modify plasma lipoproteins in the hypercholesterolemic rabbit.

The experiment was designed to study the effects of butters differing in conjugated linoleic acid (CLA) and trans 18:1 contents on lipoproteins associated with the risk of atherogenesis. New Zealand White male rabbits (9.6 weeks; 2.1 kg) were assigned for 6 or 12 weeks to three diets (n = 6 per diet) made of conventional pellets with 0.2% cholesterol and with 12% fat provided from a butter poor in trans-10 and trans-11 18:1 and in CLA (standard group), or rich in trans-10 18:1 (trans-10 18:1 group) or rich in trans-11 18:1 and in cis-9,trans-11 CLA (trans-11 18:1/CLA group). Blood samples were collected at the end of dietary treatments. Lipoproteins were separated by gradient-density ultracentrifugation. Lipid classes were determined enzymatically and apolipoproteins A-I and B by radial immunodiffusion. Mainly in the 12-week rabbits, higher plasma triglycerides and apolipoprotein B levels shown in the standard and trans-10 18:1 groups compared with those in the trans-11 18:1/CLA group are associated with higher plasma levels of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) also shown in these two groups. In the 12-week rabbits, a shift towards denser LDL, considered as more atherogenic, was shown only in the trans-10 18:1 group. In these animals, the VLDL + LDL to HDL ratio was 1.7-2.3 times higher in the trans-10 18:1 group than in the other groups (P = 0.076). These results suggest a rather neutral effect of trans-11 18:1/CLA butter towards the risk of atherogenesis, whereas trans-10 18:1 butter would tend to be detrimental.

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.

Conjugated linoleic acid isomers and their conjugated derivatives inhibit growth of human cancer cell lines.

Conjugated linoleic acid (CLA), mainly c9,t11- and t10,c12-isomers, and polyunsaturated n-3 fatty acids (n-3 PUFA) have been shown to reduce tumor growth. This study compared, on a set of human tumor cells (breast, lung, colon, prostate and melanoma), the antiproliferative effects of: i) trans monounsaturated fatty acids (MUFA) vs. cis MUFA and MUFA vs. PUFA, ii) individual isomers of CLA vs. linoleic acid, iii) CLA-conjugated derivatives vs. their non-conjugated homologues and vs. CLA isomers. Tumor cells were exposed to medium containing individual FA (100 microM) for 48 h and their proliferation was determined by measuring the cellular DNA content (fluorescent Hoechst 33342 dye). The antiproliferative effects of FA varied with the type of cells and were mainly dependent on the degree of unsaturation and on the position and configuration of their double bonds. One isomer of CLA (t9,t11-18:2) and CLA-conjugated derivatives exhibited the strongest growth-inhibitory effect against cancer cells. These results suggest that ruminant products contain active compounds against human tumor cell proliferation.

In vitro metabolism of rumenic acid in bovine liver slices.

Ruminant products are the major source of CLA for humans. However, during periods of fat mobilization, the liver might play an important role in CLA metabolism which would limit the availability of the latter for muscles and milk. In this context, rumenic acid (cis-9, trans-11 CLA) metabolism in the bovine liver (n = 5) was compared to that of oleic acid (n = 3) by using the in vitro liver slice method. Liver slices were incubated for 17 h in a medium containing 0.75 mM of FA mixture and 55 microM of either [1-(14)C] rumenic acid or [1-(14)C] oleic acid at 37 degrees C under an atmosphere of 95% O(2)-5% CO(2). Rumenic acid uptake by liver slices was twice (P = 0.009) that of oleic acid. Hepatic oxidation of both FA (> 50% of incorporated FA) led essentially to the production of acid-soluble products and to a lower extent to CO(2) production. Rumenic acid was partly converted (> 12% of incorporated rumenic acid) into conjugated C18:3. CLA and its conjugated derivatives were mainly esterified into polar lipids (71.7%), whereas oleic acid was preferentially esterified into neutral lipids (59.8%). Rumenic acid secretion as part of VLDL particles was very low and was one-fourth lower than that of oleic acid. In conclusion, rumenic acid was highly metabolized by bovine hepatocytes, especially by the oxidation pathway and by its conversion into conjugated C18:3 for which the biological properties need to be elucidated.

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 non-ruminant) 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 degrees 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 microM [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.

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.

Dietary linoleic acid-induced hypercholesterolemia and accumulation of very light HDL in steers.

This experiment was designed to study the effects in fattening steers of n-6 PUFA supplementation on the plasma distribution and chemical composition of major lipoproteins (TG-rich lipoproteins: d < 1.006 g/mL; intermediate density lipoproteins + LDL: 1.019 < d < 1.060 g/mL; light HDL: 1.060 < d < 1.091 g/mL; and heavy HDL: 1.091 < d < 1.180 g/mL). For a period of 70 d, animals [454 +/- 20 d; 528 +/- 36 kg (mean +/- SD)] were given a control diet (diet C, n = 6) consisting of hay and concentrate mixture (54 and 46% of diet dry matter, respectively) or the same diet supplemented with sunflower oil (4% of dry matter), given either as crushed seeds (diet S, n = 6) or as free oil continuously infused into the duodenum through a chronic canula to avoid ruminal PUFA hydrogenation (diet O, n = 6). Plasma lipids increased in steers given diet S (x1.4, P < 0.05) and diet O (x2.3, P < 0.05), leading to hyperphospholipemia and hypercholesterolemia. With diet S, hypercholesterolemia was associated with higher levels of light (x1.4, P < 0.05) and heavy HDL (x1.3, NS). With diet O, it was linked to higher levels of light HDL (x1.8, P < 0.005) and to very light HDL accumulation within density limits of 1.019 to 1.060 g/mL, as demonstrated by the apolipoprotein A-I profile. Diet O favored incorporation of 18:2n-6 into polar (x2.2, P < 0.05) and neutral lipids (x1.5 to x8, P < 0.05) at the expense of SFA, MUFA, and n-3 PUFA. Thus, protection of dietary PUFA against ruminal hydrogenation allowed them to accumulate in plasma lipoproteins, but the effects of hypercholesterolemia on animal health linked to very light HDL accumulation remain to be elucidated.

Linoleate supplementation in steers modifies lipid composition of plasma lipoproteins but does not alter their fluidity.

The health value for man of lipids in bovine muscles can be improved by the addition of PUFA to the animals' diets, but such treatments can modify fluidity of plasma lipoproteins and therefore their metabolic functions. The aim of the present study was to analyse whether changes in chemical composition of lipoproteins in steers fed sunflower oil-rich diets altered lipoprotein fluidity, measured by fluorescence polarization and electron spin resonance. LDL, light HDL and heavy HDL fractions were isolated by ultracentrifugation from plasma of eighteen crossbred Charolais x Salers steers. For a period of 70 d, animals were given a control diet (C, n 6) consisting of hay (540 g/kg) and concentrate mixture (460 g/kg) or the same basal diet supplemented with sunflower oil rich in n-6 PUFA (40 g/kg diet DM), given either as crushed seeds (S, n 6) or as a free oil infused directly into the duodenum (O, n 6), thus avoiding ruminal hydrogenation of PUFA. We have shown that in bovine animals: (1) fluidity measurements by fluorescence polarization must be made at the bovine physiological temperature (38.5 degrees C); (2) heavy HDL always appear as the less fluid lipoparticles; (3) electron spin resonance, which does not depend on lipoparticle size, is more appropriate to compare the fluidity of LDL with that of light HDL. The values for lipoprotein fluidity measured by both methods indicated that linoleate-rich diets did not have any effect when compared with diet C; however, chemical variables support a fluidification of lipoparticles, since in steers given the diet O, n-6 PUFA concentrations increased in polar (x1.8) and neutral (x1.6) lipids in lipoparticles (P=0.0001). The phospholipid:protein ratio increased in light (+20 %, P=0.019) and heavy (+23 %, P=0.06) HDL and especially in LDL (+46 %, P=0.0001); the total cholesterol:phospholipid ratio decreased in the three lipoprotein classes (-15 to -30 %, NS). Diet S led to similar but less pronounced effects. We concluded that linoleate-rich diets modified the chemical composition of plasma lipoproteins in steers, but did not alter their fluidity; this probably occurred as a result of 'homeoviscous adaptation', which ensured their functional capacity.

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

Hepatic metabolism of the two main isomers of CLA (9cis-11trans, 10trans-12cis C18:2) 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 degrees C for 17 h 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 microM [1-(14)C]9cis-11trans C18:2,11-(14)C]10trans-12cis C18:2, or 11-(14)C]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-12cis, 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 isomers 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 (70% of esterifled FA) and, to a lesser extent, into polar 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-12cis being 2.2-fold higher than that of 9cis-11trans (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.