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Triglyceride composition of bovine milk fat with elevated levels of linoleic acid.

The effect of increasing the linoleic acid (18:2) content of milk fat on the composition and structure of the triglycerides (TG) was investigated. Protected sunflower seed supplement was added to the diet of a cow grazing on pasture, and the structure and composition of the milk fat compared with the milk fat from its monozygous twin which had been fed a control diet. The relative proportions of TG fractions of high, medium, and low molecular weight in the milk fat with elevated levels of 18:2 (15.5% 18:2) were 43.0, 19.5, and 37.5 moles %, respectively, compared with 36.1, 19.7, and 44.2 moles %, respectively in the milk fat from the cow fed the control diet. Separation of these three TG fractions of each milk fat into TG classes with different levels of unsaturation showed that the milk fat with elevated levels of 18:2 contained higher proportions of diene, triene, and tetraene TG and correspondingly lower proportions of saturated and, to a lesser extent, monoene TG. The saturated and monoene TG from the two milk fats had similar fatty acid compositions. However, the diene TG of the 18:2-rich milk fat included high proportions of the combination of 18:2 with two saturated fatty acids (FA) which are minor constituents of normal milk fats. Likewise, the triene TG reflected the presence of 18:2 in combination with 18:1 and a saturated FA.

Animal Feed↗

Hepatic lipid and carbohydrate metabolism in rats fed a commercial mixture of conjugated linoleic acids (Clarinol G-80).

BACKGROUND: Conjugated linoleic acids (CLAs) exert numerous effects in animal models as well as in humans. Among other things, CLAs decrease plasma lipid levels and bring about hepatic steatosis. The latter effects are attributed to an agonistic action of CLAs on the peroxisome-proliferator-activated receptor family primarily responsible for activating genes involved in lipid metabolism and are related to changes in mRNA levels. Such changes are not necessarily reflected in changes in activity of controlling enzymes. AIM OF THE STUDY: To investigate the effects of CLAs treatment on lipid metabolism, we determined lipid concentrations in plasma, lipoproteins and liver and measured the activity of a number of key enzymes in hepatic lipid metabolism as differences in lipid concentrations should be related to changes in enzyme activities. These variables were determined with the rat as a model. METHODS: Rats were fed a control diet or a diet containing 1.15% trans-10, cis-12 isomer and 1.11% cis-9, trans-11 isomer as part of a commercial mixture of CLAs. After 2 w the animals were killed, and plasma and liver fractions isolated. Subsequently, lipid concentrations of cholesterol, triacylglycerols and phospholipids were determined in the isolated lipoproteins. In livers homogenates, the concentrations of glycogen, cholesterol, triacylglycerol and phospholipids and the activities of enzymes catalyzing pacesetting steps of metabolism were determined, i. e. acetyl-CoA carboxylase, fatty acid synthase, diacylglycerol acyltransferase, 3-hydroxy- acyl-CoA dehydrogenase, citrate synthase and phosphofructokinase. RESULTS: CLAs induced a lowering of the cholesterol levels in total plasma and in LDL and HDL lipoproteins and of phospholipid concentrations in LDL and HDL. CLAs treatment decreased the hepatic activity of diacylglycerol acyltransferase and had no effect on any of the other enzyme activities. CONCLUSIONS: In other studies enhanced specific activities of ACC and FAS were found in livers of mice using the same or similar methods and experimental protocol as in the present study. The lack of effect of CLAs treatment on hepatic key enzymes of fatty acid synthesis and oxidation in Wistar rats questions the use of this strain for studying the mechanism(s) underlying CLA's effects on these parameters. However, in the rat model we observed reduced levels of cholesterol in total plasma and in LDL and HDL. Therefore, some aspects like loss of body fat are better studied in mice; for other aspects like reduction in serum cholesterol level the rat may be the model of choice.

Acetyl-CoA Carboxylase↗

Linoleic acid inhibition of naturally occurring lymphocytotoxicity to breast cancer-derived cells measured by a chromium-51 release assay.

Long-chain unsaturated free fatty acids have been shown to inhibit the immune responses of lymphocytes in vivo and in transformation and macrophage inhibition factor assays in vitro. The purpose of this study was to test the effect of linoleic acid on naturally occurring lymphocytotoxicity in a 51Cr release microcytotoxicity assay with the use of a human breast carcinoma cell line (AlAb) as source of the target cells. In the presence of increasing concentrations of linoleic acid, a linear decrease in lymphocytotoxicity was observed with complete inhibition at concentrations of 0.05-0.07 mg/ml. Concentrations of linoleic acid greater than 0.07 mg/ml resulted in nonspecific toxicity to target cells but not to lymphocytes as determined by trypan blue exclusion tests. When lymphocytes were pretreated with linoleic acid, washed, and then tested for cytotoxicity in the absence of linoleic acid, suppression of lymphocytotoxicity was still demonstrated. When leukocytes were depleted of macrophages, cytotoxicity in the absence of linoleic acid was enhanced 50-100%; complete inhibition of cytotoxicity was still observed at 0.05 mg linoleic acid/ml. When B- and T-cell populations were separated, each population showed typical decreases in lymphocytotoxicity in the presence of linoleic acid; however, the B-cell fraction (containing "null" cells) was two to three times more efficient at cell killing than was the T-cell fraction.

Antigens, Neoplasm↗

Conjugated linoleic acids: are they beneficial or detrimental to health?

Conjugated linoleic acids (CLAs) comprise a family of positional and geometric isomers of linoleic acid (18:2n-6; LA) that are formed by biohydrogenation and oxidation processes in nature. The major dietary sources of these unusual fatty acids are foods derived from ruminant animals, in particular dairy products. The main form of CLA, cis-9, trans-11-18:2, can be produced directly by bacterial hydrogenation in the rumen or by delta-9 desaturation of the co-product vaccenic acid (trans-11-18:1) in most mammalian tissues including man. The second most abundant isomer of CLA is the trans-10, cis-12-18:2 form. Initially identified in grilled beef as a potential anti-carcinogen a surprising number of health benefits have subsequently been attributed to CLA mixtures and more recently to the main individual isoforms. It is also clear from recent studies that the two main isoforms can have different effects on metabolism and cell functions and can act through different cell signalling pathways. The majority of studies on body compositional effects (i.e. fat loss, lean gain), on cancer and cardiovascular disease attenuation, on insulin sensitivity and diabetes and on immune function have been conducted with a variety of animal models. Observations clearly emphasise that differences exist between mammalian species in their response to CLAs with mice being the most sensitive. Recent studies indicate that some but not all of the effects observed in animals also pertain to human volunteers. Reports of detrimental effects of CLA intake appear to be largely in mice and due mainly to the trans-10, cis-12 isomer. Suggestions of possible deleterious effects in man due to an increase in oxidative lipid products (isoprostanes) with trans-10, cis-12 CLA ingestion require substantiation. Unresponsiveness to antioxidants of these non-enzymatic oxidation products casts some doubt on their physiological relevance. Recent reports, albeit in the minority, that CLAs, particularly the trans-10, cis-12 isomer, can elicit pro-carcinogenic effects in animal models of colon and prostate cancer and can increase prostaglandin production in cells also warrant further investigation and critical evaluation in relation to the many published anti-cancer and anti-prostaglandin effects of CLAs.

Animals↗

The trans-10,cis-12 isomer of conjugated linoleic acid downregulates stearoyl-CoA desaturase 1 gene expression in 3T3-L1 adipocytes.

Conjugated linoleic acids (CLA) are a group of positional and geometric conjugated dienoic isomers of linoleic acid. The objective of this study was to determine the effects of the cis-9,trans-11 and trans-10,cis-12 isomers of conjugated linoleic acid on lipid composition and gene expression during the differentiation of mouse 3T3-L1 preadipocytes. Treatment of differentiating 3T3-L1 preadipocytes with trans-10,cis-12 conjugated linoleic acid (CLA) resulted in a dose-dependent decrease in the expression of the stearoyl-CoA desaturase 1 gene (SCD1). The expression of other adipocyte genes such as adipose P2 (aP2), fatty acid synthase (FAS), SCD2 and the key adipogenic transcription factors, peroxisome proliferator-activated receptor gamma2 (PPARgamma2) and CCAAT enhancer binding protein alpha (C/EBPalpha), remained elevated. Cells treated with trans-10,cis-12 CLA exhibited smaller lipid droplets, with reduced levels of the major monounsaturated fatty acids, palmitoleate and oleate. By contrast, the cis-9,trans-11 isomer did not alter adipocyte gene expression. Repression of the stearoyl-CoA desaturase gene expression in adipocytes by the trans-10,cis-12 isomer may contribute to the mechanisms by which CLA reduces body fat in mice.

3T3 Cells↗

Hydroperoxidase activity of lipoxygenase: a potential pathway for xenobiotic metabolism in the presence of linoleic acid.

The ability of hydroperoxidase activity of soybean lipoxygenase to co-oxidize xenobiotics was examined in the presence of linoleic acid. The guaiacol oxidation rate was proportional to the linoleic acid dioxygenation rate of the enzyme preparation. The tetraguaiacol formation was dependent upon the concentration of guaiacol, linoleic acid and the amount of enzyme. Boiled enzyme was devoid of activity suggesting enzymatic nature of the co-oxidation reaction. Besides guaiacol, benzidine, tetramethyl-p-phenylenediamine, dimethoxybenzidine, 2,2'-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid), p-phenylenediamine and tetramethylbenzidine were oxidized suggesting that the hydroperoxidase activity of lipoxygenase may be a potential pathway of xenobiotic oxidation.

Hydrogen-Ion Concentration↗

The effects of conjugated linoleic acid on human health-related outcomes.

Conjugated linoleic acid (CLA) is a collective term for a mixture of positional and geometric isomers of conjugated dienoic derivatives of linoleic acid. CLA has received considerable attention as a result of animal experiments that report anti-carcinogenic, anti-atherogenic and anti-diabetic properties, and modulation of body composition and immune function. Several studies of CLA supplementation in human subjects have now been published, but in contrast to animal studies there has been marked variation between reports on the health-related outcomes. The consensus from seventeen published studies in human subjects is that CLA does not affect body weight or body composition. Some detrimental effects of the trans-10,cis-12 CLA isomer have also been reported in terms of altered blood lipid composition and impaired insulin sensitivity. Finally, CLA has only limited effects on immune functions in man. However, there have been reports of some interesting isomer-specific effects of CLA on the blood lipid profile, but not on immune function. These isomer-specific effects need further investigation. Until more is known, CLA supplementation in man should be considered with caution.

Animals↗

A possible mechanism for the stimulation of metalloproteinase production in human aortic intimal smooth muscle cells by linoleic acid hydroperoxide.

To approach the mechanism of the stimulating effect of linoleic acid hydroperoxide on the production of matrix metalloproteinases (MMPs) in human aortic intimal smooth muscle cells (ISMC), we investigated the effect of the hydroperoxide on the cytosolic level of Ca2+. Linoleic acid hydroperoxide provoked an increase in the cytosolic Ca2+ level, but it had no effect on the level of inositol phosphates (IPs) in these cells, in contrast with the effect of platelet-derived growth factor (PDGF), which elevated the level of both Ca2+ and IPs in these cells. A23187, a calcium ionophore, stimulated ISMC to produce matrix prometalloproteinase 1. These results indicate that linoleic acid hydroperoxide stimulates the production of MMPs in ISMC by elevation of the cytosolic Ca2+ level without the intervention of IPs. In addition, we found that the hydroperoxide has no effect on the binding of PDGF to its specific receptor on ISMC.

Aorta, Thoracic↗

Mammary transfer of vitamin E in cows treated with vitamin A or linoleic acid.

The effect of an intravenous injection of vitamin A alcohol and subcutaneous injections of linoleic acid on the mammary transfer of an intravenous injection of vitamin E acetate was studied with 15 Holstein cows. The cows received either an intravenous injection of 3 g vitamin E acetate (controls), intravenous injections of 3 g vitamin E acetate and 1 million IU vitamin A alcohol, or an intravenous injection of 3 g vitamin E acetate and subcutaneous injections totaling 40 g of linoleic acid. Milk samples were at 12-h intervals, two prior to and six following treatment. The main influence of vitamin A alcohol and linoleic acid on mammary transfer of vitamin E was to delay secretion of vitamin E in milk. However, total secretion of vitamin E was not reduced by injection of either vitamin A alcohol or linoleic acid. Vitamin E injection produced substantial increases in vitamin E in milk, but less than 1% of the dose could be accounted for in the milk.

Animals↗

Dioxygenase and peroxidase activities of soybean lipoxygenase: synergistic interaction between linoleic acid and hydrogen peroxide.

The interaction of H2O2 with soyabean lipoxygenase was investigated in the presence of linoleic acid. Dioxygenase activity was significantly higher at pH 9.0 than at pH 6.0. H2O2 at concentrations less than 1.0 nM stimulated linoleic acid oxidation synergistically and the magnitude of synergism was higher at pH 9.0. Linoleic acid dependent peroxidase activity towards benzidine, guaiacol, tetramethylbenzidine (TMBD) and tetramethylphenylenediamine (TMPD) was higher at pH 9.0, whereas pyrogallol and ABTS oxidation rates were higher at pH 6.0. H2O2 supported oxidation of benzidine, guaiacol, pyrogallol and ABTS was higher at pH 6.0, whereas TMPD, TMBD exhibited higher oxidation rates at pH 9.0. H2O2 in the presence of linoleic acid produced synergism in xenobotic metabolism and depending upon the substrate in question upto 11-fold increase in oxidation rate was noted.

Drug Synergism↗

Augmented acute hypotensive effect of dihydralazine and clonidine after linoleic acid rich diet in normotensive conscious rats.

The influence of dietary linoleic acid (LA) content on the cardiovascular effects of dihydralazine and clonidine was investigated in conscious rats. Male normotensive rats were fed either a diet rich in linoleic acid (LA) (13.3 cal-% LA) or a diet deficient in LA (0.5 cal-% LA) for a period of 5 months beginning in the pregnant mothers. Dihydralazine (1 mg/kg iv) or clonidine (10 micrograms/kg iv) were injected into conscious rats and blood pressure and heart rate were studied during a 20 min investigation period. The blood pressure lowering effects were higher in the rats fed the LA rich diet than in those fed the LA deficient diet 2, 5, 10 and 20 min after dihydralazine and 5 min after clonidine injection. The increase in heart rate per 10 mmHg blood pressure reduction after dihydralazine injection was lower in the rats fed on the diet rich in LA. We assume that the change in the cardiovascular effects of dihydralazine and clonidine by dietary LA may be caused by alterations of endogenous prostaglandin biosynthesis and sympathetic activity.

Animals↗

Dietary conjugated linoleic acid reciprocally modifies ketogenesis and lipid secretion by the rat liver.

The effects of dietary conjugated linoleic acid (CLA) and linoleic acid (LA) on ketone body production and lipid secretion were compared in isolated perfused rat liver. After feeding the 1% CLA diet for 2 wk, the concentration of post-perfused liver cholesterol was significantly reduced by CLA feeding, whereas that of triacylglycerol remained unchanged. Livers from CLA-fed rats produced significantly more ketone bodies; and the ratio of beta-hydroxybutyrate to acetoacetate, an index of mitochondrial redox potential, tended to be consistently higher in the liver perfusate. Conversely, cumulative secretions of triacylglycerol and cholesterol were consistently lower in the livers of rats fed CLA, and the reduction in the latter was statistically significant. Thus dietary CLA appeared to exert its hypolipidemic effect at least in part through an enhanced beta-oxidation of fatty acids at the expense of esterification of fatty acid in the liver.

Animals↗

Replacement of linoleic acid with alpha-linolenic acid does not alter blood lipids in normolipidaemic men.

The effect of partial dietary replacement of linoleic acid (18:2n-6; linoleic acid-rich diet) with alpha-linolenic acid (18:3n-3; alpha-linolenic acid-rich diet) on plasma lipids was investigated in twenty-nine healthy young men. After a 2-week stabilization period subjects were randomly assigned to either the alpha-linolenic acid-rich diet group (n 15), receiving a mean of 10.1 g of alpha-linolenic acid and 12.1 g of linoleic acid/d, or the linoleic acid-rich diet group (n 14), receiving a mean of 1.0 g of alpha-linolenic acid and 21.0 g of linoleic acid/d, for a 6-week test period. Blood samples were taken at the commencement of the stabilization period and at the start (week 0), midpoint (week 3) and endpoint (week 6) of the test period and plasma lipids analysed. The changes occurring on the linoleic acid-rich diet and alpha-linolenic acid-rich diet were compared but no significant differences in the changes in plasma total cholesterol, LDL-cholesterol, HDL-cholesterol, the subfractions HDL2 and HDL3 or triacylglycerols were found. These results indicate that dietary replacement of linoleic acid with alpha-linolenic acid in the diet of healthy male subjects offers similar cardioprotective benefits with respect to lipid metabolism.

Adolescent↗

Hemin and hemeprotein bleaching during linoleic acid oxidation by lipoxygenases.

Hemin and hemoglobin are bleached by lipoxygenases, type 1 (from soybean) or type 2 (from platelets), during linoleic acid oxidation. This process has been found to be related to the inhibition of the lipoxygenase activity, measured as hydroperoxide generation and to produce oxodienes as well. All these parameters have been determined simultaneously from measurements of the absorbance at 234, 285, 375 and 410 nm to detect hydroperoxides, oxodienes, hemin and hemoglobin, respectively, using a diode array spectrophotometer. The inhibition of lipoxygenase activity by these pigments has been found to be competitive with linoleic acid, showing an increase of 4-7-fold of the Km value of linoleic acid in the presence of concentrations of hemin and hemoglobin as low as 0.2 and 0.02 microM, respectively, for the case of platelet lipoxygenase activity. The concentrations of hemin and of hemoglobin producing the inhibition of 50% of lipoxygenase activity are: 0.25 and 0.02 microM for the platelet isoenzyme, and 1.4 and 0.18 microM for the soybean isoenzyme, respectively. From the quenching of the intrinsic fluorescence of soybean lipoxygenase activity by hemin, we have obtained a dissociation constant of hemin-soybean lipoxygenase of 0.5 microM. The results obtained in this paper for the cooxidation process of hemin and hemoglobin by lipoxygenase can be rationalized in terms of hemin binding at or near to the catalytic center, resulting in a lesser binding of linoleic acid and an enhanced release of radicals, and pigment bleaching by radicals and lipid hydroperoxides.

Animals↗

Uptake of secondary autoxidation products of linoleic acid by the rat.

Incorporation of secondary autoxidation products (SP) of linoleic acid into the rat body was investigated. Radioactive SP was administered orally to a group of 5 rats, and excretions of radioactive substances in feces, urine and respiration were measured and compared with excretions from rats fed linoleic acid and its hydroperoxides. The SP-fed group excreted 45% and the other groups about 10% of the administered radioactivity through feces. Urinary excretion accounted for 52% of activity ingested in the SP group and less than 30% in the other groups. The 14CO2 produced in each group was about 25% of the ingested activity. Incorporation of the radioactive substances of SP into tissues and organs was measured periodically after administration of a single dose. The radioactive substances accumulated in the liver between 12-24 hr after administration and accounted for 2.6% of the total amount given, the highest level of all tissues and organs. This accumulation led to an elevation of serum transaminase activities, an increase in hepatic lipid peroxide, as determined by thiobarbituric acid test, and a slight hypertrophy of liver (1.5-fold). Therefore, absorbed SP appeared to contribute to the deleterious condition of the liver.

Animals↗

Prostaglandin H-synthase-2 is the main enzyme involved in the biosynthesis of octadecanoids from linoleic acid in human dermal fibroblasts stimulated with interleukin-1beta.

This study was focused on the characterization of the metabolism of linoleic acid by human dermal fibroblasts and the effect of interleukin-1 on the biosynthesis of octadecanoids. Dermal fibroblasts untreated and treated with recombinant IL-1beta were incubated with exogenous labeled linoleic acid. A combination of high performance liquid chromatography and gas chromatography-mass spectrometry was used as the analytic technique. We found that dermal fibroblasts convert linoleic acid mainly into 13-hydroxy-9-cis,11-trans-octadecadienoic acid (13-HODE) and 9-hydroxy-10-trans,12-cis-octadecadienoic acid (9-HODE), 13(S)-HODE and 9(R)-HODE being the predominant enantiomers. IL-1beta increased the formation of both 13-HODE and 9-HODE in a concentration-dependent manner with similar EC50 values as for prostanoid formation. This effect of IL-1beta on HODEs formation was concomitant with the expression of prostaglandin H-synthase-2. Formation of octadecanoids was inhibited in a concentration-dependent manner by acetylsalicylic acid and indomethacin. Dexamethasone, actinomycin D, and cycloheximide abolished the effect of IL-1beta on HODEs biosynthesis. Octadecanoid biosynthetic activity was associated with the microsomal fraction. Dermal fibroblasts incorporated [14C]-9-HODE and [14C]-13-HODE into phospholipids, mainly into phosphatidylcholine. IL-1beta increased significantly the esterification of 13-HODE in all glycerophospholipids, the major increase being observed in phosphatidylinositol. These results indicate that prostaglandin H-synthase-2 is the enzyme responsible for the increase in the ability to form HODEs of dermal fibroblasts stimulated with IL-1beta.

Cells, Cultured↗

Response of liver microsomal mixed-function oxidases to dietary linoleic acid levels in rats.

Responses of the activities of liver microsomal mixed-function oxidases (MFOs) induced by phenobarbital (PB) to dietary linoleic acid (LA) levels were investigated in rats. Diets varied in LA content (0, 1.8, 4.1, 7.0, or 13.8 energy%) were fed to rats for 16 days. The activities of MFOs did not change significantly with increasing the dietary LA level, where they reached a plateau even at 1.8 energy %, although the fatty acid composition of liver microsomal lipids reflected well that of dietary lipids. Accordingly, 2 energy % of LA as essential fatty acid seemed to be sufficient to supply the dietary requirement as assessed by the activities of PB-induced MFOs.

Animals↗

Linoleic acid and linolenic acid elongation products in muscle tissue of Sncerus caffer and other ruminant species.

The metabolic elongation products of both linoleic acid and linolenic acid were found in muscle tissues of Syncerus caffer and other ruminants. The acids with four double bonds were predominantly in the linoleic acid series, whereas the higher degrees of unsaturation, mainly five double bonds, were in the linolenic acid series. The total linoleic acid and linolenic acid groups were present in the relative proportions of about 4:1, in contrast with the fish oils, where the acids are mainly in the linolenic acid series. The consistent occurrence of members of both groups of acids in the animals studied here suggests to us that both may be important for structural purposes.

Animals↗