Plasmalogen metabolism-related enzymes in rat brain during aging: influence of n-3 fatty acid intake.

Plasmalogens (Pls) are phospholipids containing a vinyl-ether bond at the sn-1 position of the glycerol backbone. They represent between 1/2 and 2/3 of the ethanolamine phospholipids in the brain. During aging, the Pls content in human brain falls down. However, the role of Pls metabolism-related enzymes in the regulation of Pls levels remains to be determined. Dihydroxyacetone phosphate acyltransferase (DHAP-AT) is the enzyme involved in the first step of Pls biosynthesis. In the brain, a phospholipase A2, which selectively acts on Pls, has been isolated (Pls-PLA2s). In this work, we aimed to evaluate the impact of DHAP-AT (a key enzyme of Pls biosynthesis) and Pls-PLA2 (a specific Pls degradation enzyme) on the evolution of Pls content in the rat brain during aging. The influence of n-3 fatty acid intake was also evaluated. Littermates from two generations of n-3 deficient rats were fed an equilibrated diet containing either alpha-LNA alone or with two doses of DHA. After weaning, 3, 9 or 21 months of diet, rats were sacrificed. Enzymatic assays were performed, Pls levels were assessed and the sn-2 position of ethanolamine Pls was analyzed. DHAP-AT activity significantly increased between weaning and 3 months with a concomitant increase of brain Pls, which reached maximal levels after 9 months. Then, Pls levels and DHAP-AT activity significantly decreased while Pls-PLA2s activity significantly increased. Dietary n-3 fatty acids had no effect on DHAP-AT activity and on Pls levels. In conclusion, the increase of brain Pls content in the first part of the life may be related to the high increase of DHAP-AT activity, probably stimulated by DHA. In aged animals, the decrease of Pls levels may mainly be caused to an increase of their degradation by Pls-PLA2. Dietary DHA may not oppose the physiologic aging.

Metabolites of conjugated isomers of linoleic acid (CLA) in the rat.

Metabolism of conjugated isomers of linoleic acid (CLA) in rats was studied by feeding high quantities of CLA (180 mg/day) for six days to animals that had been reared on a fat-free diet for two weeks. After this period, animals were sacrificed and liver lipids extracted. High-performance liquid chromatography (HPLC) analyses with UV detection revealed the presence of conjugated polyunsaturated fatty acids in the total liver lipid methyl esters. After isolation by HPLC, three fatty acid metabolites were identified by gas liquid chromatography coupled with mass spectrometry as being C20:3 delta 8,12,14, C20:4 delta 5,8,12,14 and C20:4 delta 5,8,11,13. A higher quantity of C20:4 delta 5,8,12,14 compared to C20:4 delta 5,8,11,13 was observed. These must arise from the elongation and desaturation of 18:2 delta 10,12 and 18:2 delta 9,11, respectively.

Effects of aging and dietary n-3 fatty acids on rat brain phospholipids: focus on plasmalogens.

The aging brain undergoes modifications in the lipid composition of cell membranes and especially in plasmalogens. These phospholipids represent between one-half and two-thirds of the ethanolamine phospholipids in the brain. They are known to facilitate membrane fusion and act as endogenous antioxidants. During normal aging and in some pathological conditions, plasmalogen and DHA levels fall. In this context, we aimed to evaluate the influence of n-3 FA intake on plasmalogens in the brain during aging. Littermates from two generations of n-3-deficient rats were fed an n-3-deficient diet or an equilibrated diet containing either alpha-linolenic acid alone (alpha-LNA) or with two doses of DHA (0.3 or 0.6% w/w). After weaning, 9 mon of diet, or 21 mon of diet, plasmalogen levels were assessed, and the sn-2 substitutions of plasmenylethanolamines were analyzed in the cortex, striatum, and hippocampus. Our results showed that plasmalogen contents were not influenced by the diet. Plasmalogen levels were significantly decreased in aged rats compared with adults, whereas DHA levels increased in the hippocampus and remained stable in the cortex and striatum. DHA levels were significantly and similarly increased in total phospholipids and especially in plasmenylethanolamines after 9 mon of diet containing alpha-LNA alone or combined with DHA. This study showed that each structure sustained specific age-induced modifications. Dietary n-3 FA may not oppose the physiological decrease in brain plasmalogen levels during aging. Moreover, alpha-LNA appears to be equally as potent as preformed DHA at replacing DHA in the brain of our rat model.

[Short term comparative study of effects of n-9 trans-docosenoic (brassidic) acid and n-9, cis-docosenoic (erucic) acid on the cardiac lipids of weanling rats]. / Effets comparés, à très court terme, des acides n-9, trans-docosénoïque (brassidique) et n-9, cis-docosénoïque (érucique) sur les lipides cardiaques du rat sevré

Cardiac lipids (triglycerides and phospholipids) of weanling rats fed diets containing 15% by weight of rapeseed oil (RSO), hydrogenated rapeseed oil (HRSO), trierucin (TE) or tribassidin (TB) were analyzed after 3 and 7 days of feeding. The amount of C22:1 was made equal in the 4 diets. Trans-isomers of erucic acid found in HRSO and TB do not cause as high an accumulation of lipids and docosenoic acids in the heart as erucic acid. Digestibility of brassidic acid (46%) is lower than that or erucic acid (83%) but even then, the amount of brassidic acid found in cardiac lipids (calculated per gram of absorbed fatty acid) after 7 days of feeding is 10 times less than that of erucic acid. Brassidic acid, like erucic acid, can be converted into shorter monoenes (C20:1 and C18:1) since transC18:1 has been found in cardiac phospholipids of rats fed trans-docosenoic acid as the only source of trans-acid in the diet.

Gas chromatographic separation and mass spectrometric identification of mixtures of oxyphytosterol and oxycholesterol derivatives application to a phytosterol-enriched food.

Pure individual phytosterols were prepared using reversed-phase HPLC in order to obtain the oxidized compounds of sitosterol, campesterol, stigmasterol and brassicasterol. 7-Hydroxy-, 7-keto-, 5,6-epoxy-, 4beta-hydroxy-, 4-ene-6-hydroxy-, 6-keto- and 5alpha,6beta-dihydroxyphytosterols were obtained as well as analogous compounds of cholesterol. The gas chromatographic properties as well as the electronic impact mass spectra of these compounds (as trimethylsilyl ether derivatives) were studied. These data were used to identify oxyphytosterols in a spread enriched in phytosterols: the oxyphytosterols represented no more than 68 microg/g of spread (about 0.08% of phytosterols were oxidised).

Trans mono- and polyunsaturated fatty acids in human milk.

OBJECTIVE: The aim of the present study was to identify the trans isomers of C18 fatty acids in some human milk samples. SUBJECTS: Ten human milk samples from French women were collected in a local milk bank in order to assess their trans mono and polyunsaturated fatty acids (PUFA) content. INTERVENTION: The fatty acid profile was examined using methyl and isopropyl ester derivatives. The combination of gas-liquid chromatography, high-performance liquid chromatography and silver nitrate thin-layer chromatography was needed to describe the detailed fatty acid compositions, including the trans isomers of unsaturated C18 fatty acids. RESULTS: All the samples contained trans isomers of C18:1 acid (mean level 1.9 +/- 0.2% of total fatty acids), with trans vaccenic acid being the major isomer. The samples also contained various isomers of linoleic and alpha-linolenic acid, but at lower levels. Trans isomers of PUFA are the same as those present in deodorised or deep-fried oils. One sample presented an abnormally high degree of isomerisation of alpha-linolenic acid (almost 50%). This was related to the dietary habit or consuming foods that were deep-fried in rapeseed oil. This milk sample also contained some cyclic fatty acid monomers. CONCLUSION: The human milk samples collected in this study contained some trans fatty acids, including isomers of essential fatty acids. This should be taken into account in the dietary intake of the newborn.

Rapid and convenient separation of phospholipids and non phosphorus lipids from rat heart using silica cartridges.

The separation of non phosphorus lipids and phospholipids of rat heart using Sep-pack Silica cartridges is described. No cartridge preparation is necessary before utilization. The separation of lipid extracts is very fast. A complete partition of non phosphorus lipids and phospholipids is obtained.

Complete separation of phospholipids from human heart combining two HPLC methods.

The separation of phospholipid classes from human heart was achieved in two steps by high performance liquid chromatography (HPLC) using a silica column with an ultraviolet spectromonitor at 206 nm. A complete partitioning of phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylinositols (PI), phosphatidylserines (PS), cardiolipins (CL), lysophosphatidylcholines (LPC) and sphingomyelins (Sph) was obtained for further analysis.

Separation and quantification of heart and liver phospholipid classes by high-performance liquid chromatography using a new light-scattering detector.

This work describes a one-step separation of rat tissue phospholipid classes by high-performance liquid chromatography (HPLC) using a silica column and a new light-scattering detector (LSD). Complete separation of phosphatidylcholine, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylserine, sphingomyelin, lysophosphatidylethanolamine, and lysophosphatidylcholine was obtained. Direct quantification was achieved after detector calibration for each phospholipid class. The detector response was shown to be linear within the ranges used. The LSD results agreed well with those obtained by phospholipid phosphorus assay. The present method was applied to rat heart and rat liver phospholipid analysis.

Variations of trans octadecenoic acid in milk fat induced by feeding different starch-based diets to cows.

The impact of starch sources differing in their velocities of ruminal degradation on the milk fat of dairy cows was studied. The animals received diets containing a slowly degradable (potatoes) or rapidly degradable (wheat) starch concentrate (40% of the dry matter) in a total mixed diet. Milk fat was the only animal performance factor affected: Cows produced significantly less milk fat when fed the wheat diet than the potato diet (-3.3 g/kg, -122 g/d; P < 0.05). With the wheat diet, milk fat was poorer in short-chain FA and richer in unsaturated long-chain FA, especially in trans octadecenoic acid (4.4 vs. 2.7% of the total FA, P < 0.05). A very large increase in the isomer trans-10 18:1 (+1.46% of the total FA) was observed. Because no difference in volatile FA concentrations in the rumen was revealed, the increase in trans octadecenoic acids, and particularly the isomer trans-10 18:1, was associated with the larger postprandial drop in ruminal pH with wheat. Similar concentrate levels and FA profiles in both diets indicated that the decrease in milk fat was due to changes in the ruminal environment. Quicker degradation of wheat starch, and hence a greater drop in pH with this diet associated with the absence of any effect on volatile FA, strengthen the hypothesis developed in the literature of enzyme inhibition via increased levels of trans octadecenoic acids, especially the trans-10 isomer. Hence, milk fat can be decreased with rapidly degradable starch sources and not only with high levels of concentrates in the diet or added fat. More detailed work is necessary to elucidate the microorganisms involved and to determine whether metabolic pathways similar to those reported for high-concentrate diets are involved.

Effect of low levels of dietary fish oil on fatty acid desaturation and tissue fatty acids in obese and lean rats.

The effect of very low levels of dietary long-chain n-3 fatty acids on delta 6 desaturation of linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3), and on delta 5 desaturation of dihomo-gamma-linolenic acid (20:3n-6), in liver microsomes and its influence on tissue fatty acids were examined in obese and lean Zucker rats and in Wistar rats. Animals fed for 12 wk a balanced diet containing ca. 200 mg of long-chain polyunsaturated n-3 fatty acids per 100 g of diet were compared to those fed the same amount of alpha-linolenic acid. Low amounts of long-chain n-3 fatty acids greatly inhibited delta 6 desaturation of 18:2n-6 and delta 5 desaturation of 20:3n-6, while delta 6 desaturation of 18:3n-3 was not inhibited in Zucker rats and was even stimulated in Wistar rats. Inhibition of the biosynthesis of long-chain n-6 fatty acids was reflected in a decrease in arachidonic acid (20:4n-6) content of serum lipids when fasting, and also in the phospholipid fatty acids of liver microsomes. On the contrary, heart and kidney phospholipids did not develop any decrease in 20:4n-6 during fish oil ingestion. Docosahexaenoic acid (22:6n-3), present in the dietary fish oil, was increased in serum lipids and in liver microsome, heart, and kidney phospholipids.

The effect of conjugated linoleic acid isomers on fatty acid profiles of liver and adipose tissues and their conversion to isomers of 16:2 and 18:3 conjugated fatty acids in rats.

Conjugated linoleic acid (CLA) is a collective term that describes different isomers of linoleic acid with conjugated double bonds. Although the main dietary isomer is 9cis,11trans-18:2, which is present in dairy products and ruminant fat, the biological effects of CLA generally have been studied using mixtures in which the 9cis,11trans- and the 10trans,12cis-18:2 were present at similar levels. In the present work, we have studied the impact of each isomer (9cis,11 trans- and 10trans,12cis-18:2) given separately in the diet of rats for 6 wk. The 10trans,12cis-18:2 decreased the triacylglycerol content of the liver (-32%) and increased the 18:0 content at the expense of 18:1 n-9, suggesting an alteration of the delta9 desaturase activity, as was already demonstrated in vitro. This was not observed when the 9cis,11trans-18:2 was given in the diet. Moreover, the 10trans,12cis-18:2 induced an increase in the C22 polyunsaturated fatty acids in the liver lipids. The 10trans,12cis-18:2 was mainly metabolized into conjugated 16:2 and 18:3, which have been identified. The 9cis,11trans isomer was preferentially metabolized into a conjugated 20:3 isomer. Thus, the 9cis,11trans- and the 10trans,12cis-CLA isomers are metabolized differently and have distinct effects on the metabolism of polyunsaturated fatty acids in rat liver while altering liver triglyceride levels differentially.

Geometry of conjugated double bonds of CLA isomers in a commercial mixture and in their hepatic 20:4 metabolites.

Rats were fed a fat-free diet for 2 wk. After this period, while maintaining the animals on the same diet, the rats were given intragastrically 180 mg per day of a mixture of conjugated linoleic acids (CLA) as triacylglycerols. Gas chromatography mass spectrometry (GC-MS) analyses of this mixture, as well as hydrazine reduction and GC-MS and GC-Fourier transform infrared analyses of the resulting monoenes, revealed the presence of two major isomers, the 9c,11t- and the 10t,12c-18:2 accompanied by smaller amounts of the 8t, 10c and the 11c, 13t-18:2 isomers. Minor quantities of cis,cis and trans,trans conjugated isomers also were detected. The total fatty acid methyl esters from the liver lipids were fractionated by reversed-phase high-performance liquid chromatography, and the fraction containing the 20:4 isomers was further fractionated by silver nitrate thin-layer chromatography. A band containing two 20:4 conjugated isomers was submitted to hydrazine reduction and the resulting monoenes analyzed by GC-MS as dimethyl-oxazoline derivatives. The two conjugated isomers were tentatively identified as 5c,8c, 11c, 13t-20:4 and 5c,8c,12t,14c-20:4. These could be formed by desaturation and elongation of the 9c,11t- and 10t,12c-18:2 present in the commercial CLA mixture.

Effects of conjugated linoleic acid isomers on lipid-metabolizing enzymes in male rats.

Male weanling Wistar rats (n = 15), weighing 200-220 g, were allocated for 6 wk to diets containing 1% (by weight) of conjugated linoleic acid (CLA), either as the 9c,11 t-isomer, the 10t,12c-isomer, or as a mixture containing 45% of each of these isomers. The five rats of the control group received 1% of oleic acid instead. Selected enzyme activities were determined in different tissues after cellular subfractionation. None of the CLA-diet induced a hepatic peroxisome-proliferation response, as evidenced by a lack of change in the activity of some characteristic enzymes [i.e., acyl-CoA oxidase, CYP4A1, but also carnitine palmitoyltransferase-I (CPT-I)] or enzyme affected by peroxisome-proliferators (glutathione S-transferase). In addition to the liver, the activity of the rate-limiting beta-oxidation enzyme in mitochondria, CPT-I, did not change either in skeletal muscle or in heart. Conversely, its activity increased more than 30% in the control value in epididymal adipose tissue of the animals fed the CLA-diets containing the 10t,12c-isomer. Conversely, the activity of phosphatidate phosphohydrolase, a rate-limiting enzyme in glycerolipid neosynthesis, remained unchanged in adipose tissue. Kinetic studies conducted on hepatic CPT-I and peroxisomal acyl-CoA oxidase with CoA derivatives predicted a different channeling of CLA isomers through the mitochondrial or the peroxisomal oxidation pathways. In conclusion, the 10t,12c-CLA isomer seems to be more efficiently utilized by the cells than its 9c,11t homolog, though the Wistar rat species appeared to be poorly responsive to CLA diets for the effects measured.

Beta-oxidation of conjugated linoleic acid isomers and linoleic acid in rats.

To assess the oxidative metabolism of conjugated linoleic acid (CLA) isomers, rats were force-fed 1.5-2.6 MBq of [1-14C]-linoleic acid (9c,12c-18:2), -rumenic acid (9c,11t-18:2), or-10trans,12cis-18:2 (10t,12c-18:2), and 14CO2 production was monitored for 24 h. The animals were then necropsied and the radioactivity determined in different tissues. Both CLA isomers were oxidized significantly more than linoleic acid. Moreover, less radioactivity was recovered in most tissues after CLA intake than after linoleic acid intake. The substantial oxidation of CLA isomers must be considered when assessing the putative health benefits of CLA supplements.

Conjugated linolenic acid (CLnA), conjugated linoleic acid (CLA) and other biohydrogenation intermediates in plasma and milk fat of cows fed raw or extruded linseed.

Thirty lactating dairy cows were used in a 3 × 3 Latin-square design to investigate the effects of a raw or extruded blend of linseed and wheat bran (70:30) on plasma and milk fatty-acids (FA). Linseed diets, containing 16.6% linseed blend on a dry-matter basis, decreased milk yield and protein percentage. They decreased the proportions of FA with less than 18 carbons in plasma and milk and resulted in cis-9, cis-12, cis-15 18:3 proportions that were more than three and four times higher in plasma and milk, respectively, whereas cis-9, cis-12 18:2 proportions were decreased by 10-15%. The cis-9, trans-11, cis-15 18:3 isomer of conjugated linolenic acid was not detected in the milk of control cows, but was over 0.15% of total FA in the milk fat of linseed-supplemented cows. Similarly, linseed increased plasma and milk proportions of all biohydrogenation (BH) intermediates in plasma and milk, including the main isomer of conjugated linoleic acid cis-9, trans-11 18:2, except trans-4 18:1 and cis-11, trans-15 18:2 in plasma lipids. In milk fat, compared with raw linseed, extruded linseed further reduced 6:0-16:0 even-chain FA, did not significantly affect the proportions of 18:0, cis-9 18:1 and cis-9, cis-12 18:2, tended to increase cis-9, cis-12, cis-15 18:3, and resulted in an additional increase in the proportions of most BH intermediates. It was concluded that linseed addition can improve the proportion of conjugated linoleic and linolenic acids, and that extrusion further increases the proportions of intermediates of ruminal BH in milk fat.

[Comparative short-term incorporation of n-9 trans docosenoic (brassidic) and n-9 cis-docosenoic (erucic) acids into rat adipose tissue]. / Incorporation comparée à très court terme, des acides n-9 trans docosénoïque (brassidique) et n-9 cis-docosénoïque (érucique) dans le tissu adipeux du rat

Trans-isomers of erucic acid (especially brassidic acid) do not incorporate in rat adipose tissue as well as erucic acid. Brassidic acid is partially converted into shorter trans-monoenes (mainly C18:1) since these acids have been identified in adipose tissue of rats fed brassidic acid as the only source of trans-fatty acid in the diet.

[Influence of prolonged physical training on the composition of fatty acids of epididymal adipose tissue and of the carcass in the young rat on a dietary regime of sunflower, rapeseed or primor oil]. / Influence d'un entraînement physique prolongé sur la composition en acides gras du tissu adipeux épididymaire et de la carcasse chez le jeune rat soumis à un régime à base d'huile de tournesol, colza ou primor.

Weanling SPF male rats were fed for 12 weeks purified diets containing by weight 15 p. 100 sunflower oil, high erucic acid rapeseed oil or low erucic acid rapeseed oil. During the same period of time they were subjected to a moderate treadmill-running program and compared with sedentary animals fed the same diets. At the end of the experiment, epididymal and perirenal adipose tissues were removed and weighed, carcass lipids were extracted and weighed and their fatty acid compositions were determined by GLC on glass capillary columns. Training decreased body weight, fat pad weight and lipid content of the carcass. However the decrease of fat pad weight in response to exercise was more important in the case of perirenal adipose tissue than in that of epididymal adipose tissue. Fatty acid composition was greatly influenced by the nature of the dietary lipids but little affected by training. Palmitic, palmitoleic, oleic, linoleic and eicosenoic acid stores in the epididymal fat of trained rats decreased but not those of stearic, linolenic and erucic acids.

Influence of physical training on the effects of dietary oils on cardiac morphology and phospholipids in rats.

Male wistar rats fed purified diets containing 15% sunflower oil (SF) by weight, high erucic acid rapeseed oil (HEAR) or low erucic acid rapeseed oil (LEAR) for 12 weeks and subjected to a moderate treadmill running program were compared with sedentary animals fed the same diets on the basis of cardiac morphology and complete analysis of cardiac phospholipids. HEAR caused the highest incidence and number of heart lesions both in untrained and trained rats but in the latter there was a highly significant increase of the lesions. LEAR gave a higher incidence of lesions than SF in untrained rats but not in trained ones. If compared to SF, HEAR and LEAR increased cardiac diphosphatidylglycerol (DPG) and sphingomyelin (SM) content (mg/g wet tissue) and decreased phosphatidylcholines (PC) and phosphatidylethanolamines (PE) in untrained rats. Marked changes in the fatty acid pattern of these phospholipids were also observed. PE and PC in trained and untrained rats fed HEAR or LEAR contained elevated levels of C 22:5 (n-3) and C 22:6 (n-3) fatty acids whereas the C 22:4 (n-6) and C 22:6 (n-6) polyenes disappeared. Monoenes (C 18:1, C 20:1 or C 22:1) largely incorporated in DPG of rats fed the cruciferous oils. In SM levels of saturated fatty acids (C 16:0, C 18:0, C 20:0) and of n-9 monoenes (C 18:1, C 20:1, C 22:1, C 24:1) were higher in rats fed HEAR or LEAR than in those feds SF. These changes were mainly related to the high level of n-9 monoenes (oleic, eicosenoic and erucic acids) as well as to the high ratio linolenic/linoleic acid present in cruciferous oils. Physical training interacted with the effects of these dietary oils on the cardiac phospholipid composition. The relation between changes in phospholipid composition and the incidence and the number of cardiac lesions is discussed.