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An oxidized metabolite of linoleic acid increases intracellular calcium in rat adrenal glomerulosa cells.

EKODE, an epoxy-keto derivative of linoleic acid, was previously shown to stimulate aldosterone secretion in rat adrenal glomerulosa cells. In the present study, we investigated the effect of exogenous EKODE on cytosolic [Ca(2+)] increase and aimed to elucidate the mechanism involved in this process. Through the use of the fluorescent Ca(2+)-sensitive dye Fluo-4, EKODE was shown to rapidly increase intracellular [Ca(2+)] ([Ca(2+)](i)) along a bell-shaped dose-response relationship with a maximum peak at 5 microM. Experiments performed in the presence or absence of Ca(2+) revealed that this increase in [Ca(2+)](i) originated exclusively from intracellular pools. EKODE-induced [Ca(2+)](i) increase was blunted by prior application of angiotensin II, Xestospongin C, and cyclopiazonic acid, indicating that inositol trisphosphate (InsP(3))-sensitive Ca(2+) stores can be mobilized by EKODE despite the absence of InsP(3) production. Accordingly, EKODE response was not sensitive to the phospholipase C inhibitor U-73122. EKODE mobilized a Ca(2+) store included in the thapsigargin (TG)-sensitive stores, although the interaction between EKODE and TG appears complex, since EKODE added at the plateau response of TG induced a rapid drop in [Ca(2+)](i). 9-oxo-octadecadienoic acid, another oxidized derivative of linoleic acid, also increases [Ca(2+)](i), with a dose-response curve similar to EKODE. However, arachidonic and linoleic acids at 10 microM failed to increase [Ca(2+)](i) but did reduce the amplitude of the response to EKODE. It is concluded that EKODE mobilizes Ca(2+) from an InsP(3)-sensitive store and that this [Ca(2+)](i) increase is responsible for aldosterone secretion by glomerulosa cells. Similar bell-shaped dose-response curves for aldosterone and [Ca(2+)](i) increases reinforce this hypothesis.

Aldosterone↗

Lack of effect of linoleic acid on the high-density-lipoprotein-cholesterol fraction of plasma lipoproteins.

Low-fat, natural diets were fed to 11 middle-aged, healthy male subjects for two 40-d periods to determine effects on plasma lipoproteins. The diets were designed to maintain saturated and monounsaturated fatty acids at 10 en% and linoleic acid (LA) at 3.8 and 10.8 en%. When compared with baseline concentrations, total cholesterol decreased 10% at the end of the lower LA diet and 15% at the end of the higher LA diet. Low-density-lipoprotein cholesterol (LDL-C) decreased 18% and 22%, respectively, during the same periods. Plasma high-density-lipoprotein cholesterol (HDL-C) at the end of each dietary period was not significantly different but the midpoint values were lower by 12.5% on the lower LA diet and 7.3% on the higher LA diet. Apolipoprotein B decreased 27% at the end of the lower LA diet and 37% at the end of the higher LA diet whereas apolipoprotein A-I increased 18% and 24%, respectively. HDL-C was not reduced by dietary LA over a 6-wk period.

Apolipoprotein A-I↗

Metabolism of U13C-labeled linoleic acid in lactating women.

Long chain polyunsaturated fatty acids are nutritionally important constituents of breast milk. The origin of these fatty acids in milk has not been clearly identified. We studied the contribution of maternal endogenous conversion of linoleic acid to milk dihomo-gamma-linolenic and arachidonic acids, using stable isotope techniques. Six lactating women ingested 1 mg of [U-13C]linoleic acid/kg body weight in the 2nd, 6th, and 12th week of lactation. Before and at several times during a 5-day period after tracer intake, samples of breath and milk were collected and the volume of daily milk production was recorded. Nutrient intakes were assessed with dietary protocols. The estimated oxidized proportion of the ingested labeled linoleic acid did not differ significantly with duration of lactation (2nd week: 18.9+/-4.5%, 6th week: 24.0+/-3.8%, 12th week: 17.7+/-3.5%, mean +/- SE), nor did transfer into milk as linoleic acid (12.7+/-1.4%, 13.1+/-2.5%, 11.7+/-2.7%, respectively). About 0.2% of the tracer appeared in milk as dihomo-gamma-linolenic acid and 0.02% as arachidonic acid. There were no statistically significant changes with time. We estimate that about 30% of milk linoleic acid is directly transferred from the diet, whereas about 11% of milk dihomo-gamma-linolenic acid and 1.2% of milk arachidonic acid originate from direct endogenous conversion of dietary linoleic acid.

Adult↗

Conjugated linoleic acid content of milk and cheese from cows fed extruded oilseeds.

Extruded oilseeds were fed to 24 dairy cows to study the influence on the conjugated linoleic acid content of milk and cheese. Cows were fed one of three diets that contained forage and grain in a ratio of 47:53. A control diet containing 13.5% soybean meal was compared with diets containing 12% full fat extruded soybeans or 12% full fat extruded cottonseed. The control, extruded soybean, and extruded cottonseed diets contained 2.73, 4.89, and 4.56% fatty acids, respectively. Measurements were made during the last 5 wk of the 8-wk experiment. The DM intakes and 3.5% fat-corrected milk yields were higher for cows fed the extruded soybean and extruded cottonseed diets than for cows fed the control diet. A tendency for lower fat and protein contents in the milk of cows fed the extruded soybean and extruded cottonseed diets was detected. Most of the C18 fatty acids were increased in the milk and cheese when extruded soybeans and cottonseeds were fed. The conjugated linoleic acid content in milk and cheese increased a mean of 109% when full fat extruded soybeans were fed and increased 77% when cottonseeds were fed compared with the conjugated linoleic acid content when the control diet was fed. Processing the milk into cheese did not alter the conjugated linoleic acid content. The conjugated linoleic acid content of milk and cheese can be increased by the inclusion of full fat extruded soybeans and full fat extruded cottonseeds in the diets of dairy cows.

Animal Feed↗

Metabolism of linoleic acid by prostaglandin endoperoxide synthase from adult and fetal blood vessels.

Linoleic acid (18:2) is converted by prostaglandin endoperoxide synthase in particulate fractions and homogenates of fetal calf aorta to its 9- and 13-hydroperoxy metabolites. These intermediates are then either dehydrated to the corresponding oxo compounds or reduced to monohydroxy products. Alternatively, the hydroperoxyoctadecadienoic acids can be converted to epoxyhydroxyoctadecenoic acids, which are hydrolyzed to trihydroxy metabolites by epoxide hydrolases present in both particulate and cytosolic fractions from aorta. Linoleic acid (Km, 442 microM) is a much poorer substrate for prostaglandin endoperoxide synthase than is arachidonic acid (20:4) (Km, 48 microM). However, the oxygenation of 18:2 by particulate fractions from aorta is linear with time for at least 5 min, whereas the oxygenation of 20:4 is linear for only 15 s. Arachidonic acid strongly inhibits the conversion of 18:2 to monohydroxy (ID50, 10 microM) and trihydroxy (ID50, 140 microM) products. Linoleic acid has a similar, but much weaker effect on the formation of 6-oxoprostaglandin F1 alpha from 20:4. Substantial amounts of both the monohydroxy (9-hydroxy-10, 12-octadecadienoic acid and 13-hydroxy-9,11-octadecadienoic acid) and trihydroxy (9,10,11-trihydroxy-12-octadecenoic acid, 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid) metabolites of 18:2 were shown by gas chromatography-mass spectrometry to be formed from endogenous substrate during incubation of slices of fetal calf aorta in physiological medium. This raises the possibility that some of these products or their hydroperoxy precursors may have some biological significance.

Aging↗

Inclusion complex of conjugated linoleic acid (CLA) with cyclodextrins.

Conjugated linoleic acid (CLA) inclusion complexes with alpha-cyclodextrin (alpha-CD), beta-cyclodextrin (beta-CD), and gamma-cyclodextrin (gamma-CD) (designated CLA/CDs inclusion complexes) were prepared to determine the mole ratio of CLA complexed with CDs and the oxidative stability of CLA in the CLA/CDs inclusion complexes. When measured by GC, (1)H NMR, and T(1) value analyses, 1 mole of CLA was complexed with 5 mol of alpha-CD, 4 mol of beta-CD, and 2 mol of gamma-CD. The oxidation of CLA induced at 35 degrees C for 80 h was completely prevented by the formation of CLA/CDs inclusion complexes.

Chromatography, Gas↗

Inhibition of hepatic stearoyl-CoA desaturase activity by trans-10, cis-12 conjugated linoleic acid and its derivatives.

Conjugated linoleic acid (CLA) has been reported to decrease stearoyl-CoA desaturase (SCD) activity by decreasing mRNA expression. This investigation was designed to determine whether structurally related compounds of CLA have a direct inhibitory effect on SCD activity. Trans-10,cis-12 CLA had strong inhibitory activity on SCD while cis-9,trans-11, and trans-9,trans-11 isomers had no effect. Trans-10 octadecenoate was not inhibitory, whereas cis-12 octadecenate was inhibitory, but not as effective as trans-10,cis-12 CLA. Of the oxygenated derivatives, 9-peroxy-cis/trans-10, trans-12 octadecadienoate was a more effective inhibitor than trans-10,cis-12 CLA, whereas 9-hydroxy-trans-10, cis-12 octadecadienoate was less effective. Interestingly, cis-11 octadecadienoate and cis-12 octadecen-10-ynoate were slightly inhibitory. However, trans-9 and trans-11 octadecenoates, and trans-9,cis-12 octadecadienoate were all inactive under test condition, as were linoleate, oleate, and arachidonate. Derivatives of CLA acid modified to alcohol, amide or chloride were all inactive. A cis-12 double bond appears to be a key structural feature for inhibiting SCD activity, especially when coupled with a trans-10 double, whereas a cis-11 double bond is less effective.

Animals↗

Detection and characterization by mass spectrometry of radical adducts produced by linoleic acid oxidation.

The formation of linoleic acid radical species under the oxidative conditions of the Fenton reaction (using hydrogen peroxide and Fe (II)) was monitored by FAB-MS and ES-MS using the spin trap 5,5-dimethyl-1-pyrrolidine-N-oxide, DMPO. Both the FAB and ES mass spectra were very similar and showed the presence of ions corresponding to carbon- and oxygen centered spin adducts (DMPO/L*, DMPO/LO*, and DMPO/LOO*). Cyclic structures, formed between the DMPO oxygen and the neighboring carbon of the fatty acid, were also observed. Electrospray tandem mass spectrometry of these ions was performed to confirm the proposed structure of these adducts. All MS/MS spectra showed an ion at m/z 114, correspondent to the [DMPO + H]+, and a fragment ion due to loss of DMPO (loss of 113 Da), confirming that they are DMPO adducts. ES-MS/MS spectra of alkoxyl radical adducts (DMPO/LO*) showed an additional ion at m/z 130 [DMPO - O + H]+, while ES MS/MS of peroxyl radical adducts (DMPO/LOO*) showed a fragment ion at m/z 146 [DMPO - OO + H]+, confirming both structures. Other fragment ions were observed, such as alkyl acylium radical ions, formed by cleavage of the alkyl chain after loss of water and the DMPO molecule. The identification of fragment ions observed in the MS/MS spectra of the different DMPO adducts suggests the occurrence of structural isomers containing the DMPO moiety both at C9 and C13. The use of ES tandem mass spectrometry, associated with spin trapping experiments, has been shown to be a valuable tool for the structural characterization of carbon and oxygen-centered spin adducts of lipid radicals.

Free Radicals↗

Conjugated linoleic acid changes swine performance and carcass composition.

Conjugated linoleic acid (CLA) is a collective term for positional and geometric isomers of linoleic acid. Dietary CLA has been shown to improve feed efficiency, decrease body fat, and increase lean tissue in laboratory animals. We hypothesized that CLA would improve performance and carcass composition and would be deposited in pork tissues. Diets of 40 crossbred pigs were supplemented with CLA to determine its effects on performance and carcass composition. Eight replications of five littermate barrows with an initial average weight of 26.3 kg were allotted at random to individual pens. Within replication dietary treatments containing 0, 0.12, 0.25, 0.5, or 1.0% CLA were assigned at random. Pigs were weighed and feed disappearance was determined at 14-d intervals. Average daily gain increased linearly as the level of CLA increased in the diet (P < 0.05). Average daily feed intake was not affected by the concentration of CLA in the diet. Therefore, a linear increase in gain:feed ratio (P < 0.05) was observed. Carcasses from animals fed control diets had greater 10th rib backfat than carcasses from animals fed CLA (P < 0.05). Ultrasound measurement and carcass measurements showed less fat depth over the loin eye at the 10th rib of pigs fed doses of CLA (P < 0.05) than that observed for control pigs. Belly hardness (firmness) increased linearly as the concentration of CLA in the diet increased when bellies were measured for firmness either lean side up (P < 0.001) or lean side down (P < 0.05). Loin dissection data demonstrated that CLA produced a quadratic treatment effect both for less intermuscular fat (P < 0.001) and less subcutaneous fat (P < 0.05) and a linear increase for bone (P < 0.05), although finished loin weight only tended to increase (P = 0.08). The CLA concentration increased in a linear relationship in both subcutaneous fat (P < 0.001) and lean tissue (P < 0.001). Dietary CLA was incorporated into pig tissues and had positive effects on performance and body composition.

Animal Feed↗

Intestinal absorption of linoleic acid in experimental renal failure.

Linoleic acid (LA) transport in rats with experimental short-term and long-term renal failure (RF) was compared with that of sham-operated normal animals on liberal food intake and pair-fed animals. The perfusions in vivo and incubations in vitro were conducted using a micellar solution containing a wide range of LA concentrations. Both absorption in vivo and uptake in vitro of LA were significantly reduced in animals with short-term RF. Lipid extraction and separation by thin-layer chromatography revealed a marked LA trapping as trilinolein (TL) in the perfused intestinal tissue in the short-term RF group. The esterification process, as defined by the rate of LA incorporation into TL, was moderately reduced in short-term RF animals. The thickness of the unstirred water layer showed no significant difference among the groups studied. In contrast, animals with long-term RF exhibited normal absorption of LA in vivo at all concentrations tested. In conclusion, LA absorption is reduced in short-term RF and restored in long-term RF. Several steps including LA transport into and TL transport out of the enterocyte and the esterification process were impaired in short-term RF. These changes are not due to alteration in the unstirred water layer, anorexia, weight loss or a rapid effect of uraemic chemical environment or circulatory factors.

Animals↗

Lymphatic delivery and in vitro pancreatic lipase hydrolysis of glycerol esters of conjugated linoleic acids in rats.

We examined the intestinal delivery of conjugated linoleic acids (CLA) given in their triacylglycerol form in the mesenteric lymph of rats. Emulsions containing a mixture of the trilinolein/triester of CLA (9:1) and a tri-[1-(14)C]-linoleyl-sn-glycerol tracer were administered by force-feeding. Lymph was collected over two time periods (0-6 and 6-24 h), and the apparent recovery of CLA was determined relative to that of [1-(14)C]-18:2(n-6). A mixture of CLA-triester/trilinolein (1:9), trilinolein or CLA-triester was separately subjected to pancreatic lipase hydrolysis in vitro to determine whether the lymphatic recovery of CLA was correlated with the initial step of digestion. Lymphatic recovery of CLA was similar to that of 18:2(n-6) (95.6+/-9.0% of the linoleic acid recovery), and isomer repartition was similar in lymph and in the oil fed, indicating that all the CLA isomers were equally absorbed by the enterocytes. Unexpectedly, the in vitro release of CLA into the absorbable forms (free fatty acids and 2-monoacyl-sn-glycerol) was consistently lower than that of 18:2(n-6). Moreover, the 9c, 11t-isomer of CLA was also released faster into the absorbable forms than its 10t,12c homolog (P = 0.05). We cannot ascribe a distinct cellular accumulation or a difference in the biological effects of different CLA isomers on the ground of a selective intestinal absorbability. Also, the physiological conditions prevailing in vivo in the digestive tract are likely to overcome the relative resistance of CLA ester bonds to pancreatic lipase hydrolysis and allow a lymphatic recovery of CLA similar to that of linoleic acid.

Analysis of Variance↗

[Conjugated linoleic acid lowers hypercholesterolemia but increases the risk for biliary lithiasis].

The term conjugated linoleic acid (CLA) refers to a series of linoleic acid isomers present in meat and diary products from ruminants that have their double bonds in a conjugated position. The aim of the present work was to study the effects of a CLA isomer, trans-10,cis-12, on cholesterolemia and biliary lithiasis risk in an animal model of diet-induced hypercholesterolemia. For that, two groups of hamsters were fed with a hypercholesterolemic diet supplemented with 0.5% linoleic acid or with the trans-10,cis-12 CLA isomer, respectively. Daily food intake and weight were determined and, 6 weeks later, serum and bile samples were obtained, and livers and spleens were dissected and weighted. Cholesterolemia, hepatic and splenic cholesterol content, and biliary cholesterol phosnolipid and bileacid concentrations were determined; Biliary Lithogenic Index was calculated, and presence of gallstones was assessed. CLA did not modify energetic intake or final body weight, spleen size or spleen cholesterol content, but it did significantly reduce total serum cholesterol (-18%) at the expense of c-LDL (-66%), and it also significantly reduced hepatic content of free cholesterol (-26%), without changes in esterified cholesterol. Besides, CLA produced a 32% increase in biliary cholesterol concentration, a 28% increase in Lithogenic Index, and a higher incidence of biliary lithiasis. Therefore, the present study shows that the CLA trans-10,cis-12 isomer is hypercholesterolemic since it increases, at least in part, cholesterol secretion to the bile. As a consequence, this effect increases the risk for biliary lithiasis.

Animals↗

Effects of linoleic acid on capping, lectin mediated mitogenesis, surface antigen expression, and fluorescent polarization in lymphocytes and BHK cells.

The incubation of linoleic acid with cells causes profound effects on membrane asociated phenomenon. Using the fluorescent probe diphenyl hexatriene (DPH) to monitor lipid changes in the microenvironment of the cell surface, we find that linoleic acid reduces the polarization values (P) in mouse lymphocytes and BHK cells. Measurements on lipids extracted from the cells grown in linoleic acid produce similar results. We also find in the mouse lymphocyte that capping of Ig is inhibited and con A stimulated mitogenesis is unaffected. In contrast to the latter effect, LPS and PHA stimulated mitogenesis is inhibited and in the rat lymph node, con A stimulated mitogenesis, greatly enhanced. We also show that linoleic acid alters the binding of antibodies to the cell surface of EL-4 lymphoma cells. These observations suggest that linoleic acid alters cellular function by interfering with protein/lipid interactions within the surface membrane.

Animals↗

Concentrations of conjugated linoleic acid (cis-9, trans-11-octadecadienoic acid) are not increased in tissue lipids of cattle fed a high-concentrate diet supplemented with soybean oil.

Conjugated linoleic acid (CLA), a mixture of isomers of linoleic acid, has many beneficial effects, including decreased tumor growth in animal cancer models. The cis-9, trans-11 isomer of CLA (CLA9,11) can be formed in the rumen as an intermediate in biohydrogenation of linoleic acid. Recent data, however, indicate that tissue desaturation of trans-fatty acids is an important source of CLA9,11 in milk. Our objective was to determine whether supplementing a high-corn diet with soybean oil (SBO; a source of linoleic acid) would increase concentrations of CLA in ruminal contents and tissue lipids. Four ruminally cannulated steers were utilized in a Latin square design with 28-d periods. A control diet (80% cracked corn, 2.0% corn steep liquor, 8.0% ground corn cobs, and 10% supplement [soybean meal, ground shelled corn, minerals, and vitamins]) was supplemented with 2.5, 5.0, or 7.5% (DM basis) SBO. Supplemental SBO did not affect ruminal pH or concentrations of the major VFA. The proportion and amount (mg FA/g DM ruminal contents) of CLA9,11 were not increased by increasing dietary SBO. However, the proportion and amount of the trans-10, cis-12 CLA isomer (CLA10,12) in ruminal contents increased linearly (P < 0.006) as dietary SBO increased. Trans-18:1 isomers in ruminal contents increased linearly (P < 0.02) as dietary SBO increased. The proportion of CLA10,12 was correlated positively (P < 0.001) with proportions of trans-C 18:1 isomers in ruminal contents. Conversely, CLA9,11 was correlated negatively (P < 0.05) with the proportions of trans-18:1 in ruminal contents. The same high-corn diet, supplemented with 0 or 5% SBO, was fed to 20 Angus-Wagyu heifers for 102 d in a randomized complete block design to determine the effect of added SBO on tissue deposition of CLA. Supplemental SBO did not affect feed intake, gain:feed, or carcass quality. Tissue samples were obtained from the hindquarter, loin, forequarter, liver, large and small intestine, and subcutaneous, mesenteric, and perirenal adipose depots. The concentration of CLA9,11 was greatest in subcutaneous adipose tissue but was not affected in any tissue by SBO. Supplementing high-corn diets with SBO does not increase CLA9,11 concentrations in tissues of fattening heifers. Research is needed to identify regulatory factors for pathways of biohydrogenation that lead to increased concentrations of CLA10,12 in ruminal contents when high-oil, high-concentrate diets are fed.

Adipose Tissue↗

Influence of dietary linoleic acid on leucocyte sodium transport and blood pressure.

In a randomised double blind study to determine whether an increase in the polyunsaturated fat linoleic acid might influence leucocyte membrane sodium transport 22 normotensive volunteers received an oral supplement of linoleic acid or placebo daily for four weeks. Mean total sodium efflux rose significantly during supplementation with linoleic acid compared with placebo. In addition, all components of lying and standing blood pressure fell, though only the fall in supine systolic pressure was significant. Dietary supplementation with linoleic acid may alter ion fluxes across the cell membrane, presumably through changes in its physicochemical structure. In addition, the change in fat intake may lower blood pressure, though to only a very modest extent.

Biological Transport↗

Evaluation of microbial strains for linoleic acid hydroxylation and reclassification of strain ALA2.

In previous studies, a new microbial strain ALA2 was isolated which produced many new products from linoleic acid [Gardner H.W., Hou C.T., Weisleder D. and Brown W. 2000. Lipids 35: 1055-1060; Hou C.T. 1998. 12,13,17-Trihydroxy-9(Z)-Octodecenoic acid and derivatives and microbial isolate for production of the acid. US Patent No. 5, 852, 196]. Strain ALA2 was preliminary identified as Clavibacter sp. based on its physiological and fatty acid profiles. To determine if strain ALA2 is the optimal strain for industrial applications, other related strains were screened for their abilities to convert linoleic acids. Two strains from Clavibacter and 20 type strains from the phylogenetically related genus Microbacterium were studied. Surprisingly, all of these strains tested showed very little or no activity in converting linoleic acid. On reexamination of the identification of strain ALA2, the sequence of the 16S ribosomal RNA gene of ALA2 was found to be 99% identical to that of Bacillus megaterium and the strain was also found to have 76.3% DNA homology to the B. megaterium type strain. Therefore, strain ALA2 is now reclassified as B. megaterium. Screening of 56 strains of B megaterium strains showed that many of them were able to produce reasonable amounts of hydroxyl fatty acids from linoleic acid, although strain ALA2 possessed the greatest activity.

Actinomycetales↗

Dietary conjugated linoleic acid alters hepatic lipid content and fatty acid composition in broiler chickens.

We examined the effect of dietary conjugated linoleic acid (CLA) on growth performance and liver composition in broiler chickens. Day-old male broiler chicks were assigned to receive a diet supplemented with corn oil (5%; n = 48) or CLA (5%; n = 48) for 21 d. Broilers fed CLA weighed less and grew at slower rates than broilers fed corn oil. Feed intake and feed conversion were higher for the corn-oil group than for the CLA dietary group. Hepatic lipid and triacylglycerol concentrations were significantly reduced by dietary CLA. The proportions of saturated fatty acids (SFA) in liver lipids increased, whereas those of monounsaturated fatty acids (MUFA) decreased with CLA supplementation. Although the total concentration of polyunsaturated fatty acids (PUFA) did not change with dietary treatment, the concentration of linoleic acid as a percentage of total methylated fatty acids decreased, and that of linolenic add increased in broilers fed CLA. The concentration of CLA isomers in liver lipids increased substantially with CLA feeding. The relative proportion of the c9,t11 CLA isomer in hepatic lipids was much higher than that of the t10,c12 or t9,t11 CLA isomers. These studies provide evidence that feeding CLA to broilers results in substantial reduction in liver fat accumulation and promotes CLA incorporation into hepatic lipid pools.

Animal Nutritional Physiological Phenomena↗