Pierre Juanéda

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 phospholipid is obtained.

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 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,11t-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 β-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.

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,11trans- 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∶1n−9, suggesting an alteration of the Δ9 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.

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.

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.

Utilisation of reversed-phase high-performance liquid chromatography as an alternative to silver-ion chromatography for the separation of cis- and trans-C18:1 fatty acid isomers

Silver ion-high-performance liquid chromatography (HPLC) has been commonly used for the separation and the analysis of trans-18:1 isomers in partially hydrogenated oils and milk fat. This paper describes an easy HPLC method using two reversed-phase columns. The cis- and trans-18:1 fatty acids isomers as methyl esters were eluted as two separate fractions. The collected fractions were analysed by gas chromatography (GC). The purity of the two fractions were tested by GC-MS and GC-Fourier transform IR.

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.

ApoB100,LDLR−/− Mice Exhibit Reduced Electroretinographic Response and Cholesteryl Esters Deposits in the Retina

PURPOSE To evaluate the retinal phenotype of 7- and 14-month-old apoB100,LDLR-/- mice, a relevant animal model of lipid metabolism dysfunction. METHODS Single-flash electroretinograms were obtained from 7- and 14-month-old apoB100,LDLR-/- and control mice fed a standard diet under both scotopic and photopic conditions. Visual cycle retinoids were analyzed in eyes from dark-adapted mice. Retinal and choroidal vascularization was evaluated with scanning laser ophthalmoscopy. Fatty acids were analyzed in the retina. Esterified and free cholesterol was detected in eye cryosections. RESULTS Scotopic and photopic b-wave amplitudes were significantly reduced in apoB100,LDLR-/- mice compared with control mice at 7 and 14 months of age (between -25% and -35% in 7-month-old animals and between -50% and -60% in 14-month-old animals at 25 cds/m2). Esterified cholesterol was found to accumulate at the basement of the retinal pigment epithelium in apoB100,LDLR-/- mouse eyes. On the contrary, no significant changes in the retinal profile of fatty acids and visual retinoids were observed in apoB100,LDLR-/- mice compared with control animals. CONCLUSIONS The exclusive expression of apoB100 in LDL receptor-null mouse altered the ERG profile, without modifying the visual cycle of retinoids and led to cholesterol deposition in the retina. These findings clearly suggest the role of cholesterol metabolism in the functioning of the retina and possibly in the etiology of ocular diseases, including age-related macular degeneration.

Occurrence of N-3 trans polyunsaturated fatty acids in human platelets

Abstract Human platelets fatty acid profiles were studied with particular interest to isomers of eicosapentaenoic and docosahexaenoic acids. C20:5 Δ 17 trans isomer was detected in most of the samples (up to 0.48% of total fatty acids). Some samples also contained the Δ 19 trans isomer of docosahexaenoic acid (0.05% max.).