bluuebunny/crossref_metadata_2025 huggingface dataset:
Response of plasma lipids to dietary cholesterol and wine polyphenols in rats fed polyunsaturated fat diets
Authors: Frémont Lucie , Gozzelino Marie T. , Linard Alain
Published: November 2006

This experiment was designed to evaluate the effects of dietary red wine phenolic compounds (WP) and cholesterol on lipid oxidation and transport in rats. For 5 wk, weanling rats were fed polyunsaturated fat diets (n−6/n−3=6.4) supplemented or not supplemented with either 3 g/kg diet of cholesterol, 5 g/kg diet of WP, or both. The concentrations of triacylglycerols (TAG, P<0.01) and cholesterol (P<0.0002) were reduced in fasting plasma of rats fed cholesterol despite the cholesterol enrichment of very low density lipoprotein + low density lipoprotein (VLDL+LDL). The response was due to the much lower plasma concentration of high density lipoprotein (HDL) (−35%, P<0.0001). In contrast, TAG and cholesteryl ester (CE) accumulated in liver (+120 and +450%, respectively, P<0.0001). However, the cholesterol content of liver microsomes was not affected. Dietary cholesterol altered the distribution of fatty acids mainly by reducing the ratio of arachidonic acid to linoleic acid (P<0.0001) in plasma VLDL+LDL (−35%) and HDL (−42%) and in liver TAG (−42%), CE (−78%), and phospholipids (−28%). Dietary WP had little or no effect on these variables. On the other hand, dietary cholesterol lowered the α‐tocopherol concentration in VLDL+LDL (−40%, P<0.003) and in microsomes (−60%, P<0.0001). In contrast, dietary WP increased the concentration in microsomes (+21%, P<0.0001), but had no effect on the concentration in VLDL+LDL. Cholesterol feeding decreased (P<0.006) whereas WP feeding increased (P<0.0001) the resistance of VLDL+LDL to copper‐induced oxidation. The production of conjugated dienes after 25 h of oxidation ranged between 650 (WP without cholesterol) and 2,560 (cholesterol without WP) μmol/g VLDL+LDL protein. These findings show that dietary WP were absorbed at sufficient levels to contribute to the protection of polyunsaturated fatty acids in plasma and membranes. They could also reduce the consumption of α‐tocopherol and endogenous antioxidants. The responses suggest that, in humans, these substances may be beneficial by reducing the deleterious effects of a dietary overload of cholesterol.
Secretion of hepatic lipase by perfused liver and isolated hepatocytes
Authors: Galan Xavier , Robert Monique Q. , Llobera Miquel , Ramírez Ignasi
Published: November 2006

Hepatic lipase is found in liver and in adrenal glands and ovaries. Because in adult rats, neither adrenals nor ovaries synthesize this enzyme, it is assumed that the liver is the origin of their hepatic lipase. Our aim was to study the secretion of hepatic lipase by the liver. We observed that plasma of both fed and fasted rats contained hepatic lipase activity. This activity was significantly correlated with that in the liver. Isolated livers, perfused with heparin‐free medium, secreted fully active hepatic lipase to the perfusate. The addition of heparin resulted in a rapid and larger release of hepatic lipase to the perfusate. In isolated hepatocytes, heparin did not affect the secretion of hepatic lipase mass, although it increased the stability of the enzyme activity. To study the degradation of hepatic lipase by hepatocytes, protein synthesis was blocked with cycloheximide, and both secreted and intracellular hepatic lipases were analyzed by Western blotting. We observed that the amount of hepatic lipase secreted equaled the decrease of intracellular mass. The total mass of the enzyme (inside and outside the cells) remained constant, at least for 90 min. In the next experiment, 0.7 nM 125I‐hepatic lipase was added to hepatocyte suspensions, and the appearance of trichloracetic acid‐soluble products was analyzed. Only 12% of the radioactivity added was associated with the cells after 90 min of incubation, and less than 2% of the hepatic lipase added was degraded. Although the association was decreased in the presence of heparin, the amount of 125I‐hepatic lipase degraded was not affected. Taking all these results into account, we propose a model for the continuous secretion of hepatic lipase by the liver.
Study of individual trans‐ and cis‐16∶1 isomers in cow, goat, and ewe cheese fats by gas‐liquid chromatography with emphasis on the trans‐Δ3 isomer
Authors: Destaillats Frédéric , Wolff Robert L. , Precht Dietz , Molkentin Joachim
Published: November 2006

Low‐temperature gas‐liquid chromatography (GLC) was applied to study the distribution profiles of isomeric trans‐and cis‐hexadecenoic acids in ruminant (cow, goat, and ewe) milk fat after their fractionation by argentation thin‐layer chromatography (Ag‐TLC). The fat was extracted from cheeses (12 samples of each species), the most common foods made with goat and ewe milks. The predominant trans‐16∶1 isomer is palmitelaidic acid (the Δ9 isomer), but it does not exceed one‐third of the total group, which itself represents 0.17% (cow), 0.16% (goat), and 0.26% (ewe) of the total fatty acids. The trans‐Δ3 16∶1 isomer, which is reported for the first time in ruminant lipids and which likely comes from the animals' feed, is present at a level of ca. 10% of the trans‐16∶1 acid group. Otherwise, all isomers with their ethylenic bond between positions Δ4 and Δ14 are observed in the three species studied, roughly showing the same relative distribution pattern. Quantitatively, the trans‐16∶1 isomers only represent ca. 5% of the sum of the trans‐16∶1 plus trans‐18∶1 isomers, and they appear of little importance in comparison. It is inferred from this and recent studies that some previously reported data that were established for consumption assessments dealt in fact mainly with iso‐17∶0 acid, which was confused with (and added to) trans‐Δ9 (palmitelaidic) acid; consequently, these results were large overestimates. Regarding the cis‐16∶1 acids, the Δ9 isomer is the prominent constituent as expected, but the second‐most important isomer is the Δ13 isomer. It does not appear that trans‐16∶1 isomers are from ruminant milk fats of great nutritional importance as compared with trans‐18∶1 isomeric acids. As for trans‐18∶1 isomers, the combination Ag‐TLC/GLC is a necessary procedure to quantitate trans‐16∶1 acids accurately and reliably. Ag‐TLC allows removal of interfering branched 17∶0 acids and cis‐16∶1 acids, and low‐temperature GLC permits an accurate measurement of all individual isomers most of which with baseline resolution.
Determination of the phospholipid/lipophilic compounds ratio in liposomes by thin‐layer chromatography scanning densitometry
Authors: Rodríguez S. , Cesio M. V. , Heinzen H. , Moyna P.
Published: November 2006

The determination of the ratio of phospholipid/ lipophilic compounds in liposomes was achieved after thin‐layer chromatography (TLC) by measuring the spot intensities of dipalmitoyl phosphatidylcholine and the lipophilic compound. The liposome components under study were separated on one TLC plate, developed in two steps, and detected after charring the plate with specific visualization reagents. The method shows good reproducibility and provides a simple way to quantify the level of lipophilic compound incorporated in the liposome bilayer.
Antioxidant reactions of α‐tocopherolhydroquinone
Authors: Liebler Daniel C. , Burr Jeanne A.
Published: November 2006

α‐Tocopherolhydroquinone (TQH2) is a product of α‐tocopherol oxidation/reduction that exerts antioxidant effects in biological systems. TQH2 inhibited autoxidation of methyl linoleate initiated by peroxyl radicals derived from thermolysis of 2,2′‐azobis(2,4‐dimethylvaleronitrile) in acetonitrile. TQH2 oxidation yielded α‐tocopherolquinone (TQ) as a major product and 2,3‐epoxy‐α‐tocopherolquinone and 5,6‐epoxy‐α‐tocopherolquinone as minor products. Each TQH2 consumed approximately two peroxyl radicals in the course of the oxidation. The data suggest that TQH2 scavenges peroxyl radicals primarily by electron transfer to form TQ and secondarily by addition‐elimination to form the epoxyquinones.
Biotransformation of linoleic acid by Clavibacter sp. ALA2: Heterocyclic and heterobicyclic fatty acids
Authors: Gardner Harold W. , Hou Ching T. , Weisleder David , Brown Wanda
Published: November 2006

Clavibacter sp. ALA2 transformed linoleic acid into a variety of oxylipins. In previous work, three novel fatty acids were identified, (9Z)‐12,13,17‐trihydroxy‐9‐octadecenoic acid and two tetrahydrofuran‐(di)hydroxy fatty acids. In this report, we confirm the structures of the tetrahydrofuran‐(di)hydroxy fatty acids by nuclear magnetic resonance as (9Z)‐12‐hydroxy‐13,16‐epoxy‐9‐octadecenoic acid and (9Z)‐7,12‐dihydroxy‐13,16‐epoxy‐9‐octadecenoic acid. Three other products of the biotransformation were identified as novel heterobicyclic fatty acids, (9Z)‐12,17;13,17‐diepoxy‐9‐octadecenoic acid, (9Z)‐7‐hydroxy‐12,17;13,17‐diepoxy‐9‐octadecenoic acid, and (9Z)‐12,17;13,17‐diepoxy‐16‐hydroxy‐9‐octadecenoic acid. Thus, Clavibacter ALA2 effectively oxidized linoleic acid at C‐7,‐12,‐13,‐16, and/or‐17.
Dietary conjugated linoleic acid did not alter immune status in young healthy women
Authors: Kelley D. S. , Taylor P. C. , Rudolph I. L. , Benito P. , Nelson G. J. , Mackey B. E. , Erickson K. L.
Published: November 2006

The purpose of this study was to examine whether conjugated linoleic acid (CLA) supplementation in human diets would enhance indices of immune status as reported by others for animal models. Seventeen women, 20–41 yr, participated in a 93‐d study conducted in two cohorts of 9 and 8 women at the Metabolic Research Unit of Western Human Nutrition Research Center. Seven subjects were fed the basal diet (19, 30 and 51% energy from protein, fat, and carbohydrate, respectively) throughout the study. The remaining 10 subjects were fed the basal diet for the first 30 d, followed by 3.9 g CLA (Tonalin)/d for the next 63 d. CLA made up 65% of the fatty acids in the Tonalin capsules, with the following isomeric composition: t10, c12, 22.6%; c11, t13, 23.6%; c9, t11, 17.6%; t8, c10, 16.6%; and other isomers 19.6%. Most indices of immune response were tested at weekly intervals, three times at the end of each period (stabilization/intervention); delayed‐type hypersensitivity (DTH) to a panel of six recall antigens was tested on study day 30 and 90; all subjects were immunized on study day 65 with an influenza vaccine, and antibody titers were examined in the sera collected on day 65 and 92. None of the indices of immune status tested (number of circulating white blood cells, granulocytes, monocytes, lymphocytes, and their subsets, lymphocytes proliferation in response to phytohemagglutinin, and influenza vaccine, serum influenza antibody titers, and DTH response) were altered during the study in either dietary group. Thus, in contrast to the reports with animal models, CLA feeding to young healthy women did not alter any of the indices of immune status tested. These data suggest that short‐term CLA supplementation in healthy volunteers is safe, but it does not have any added benefit to their immune status.
Cu2+‐induced low density lipoprotein peroxidation is dependent on the initial O2 concentration: An O2 consumption study
Authors: Lodge John K. , Traber Maret G. , Sadler Peter J.
Published: November 2006

Atherosclerotic plaques form in the arterial intima, where low density lipoprotein (LDL) is thought to be oxidatively modified at sites which may contain catalytic amounts of copper in the presence of low O2 tension. We have investigated O2 consumption during LDL peroxidation induced by Cu2+ ions in vitro and found two phases: a lag phase followed by a phase of rapid O2 consumption. The length of the lag phase was dependent on Cu2+ and on initial O2 concentrations; increasing either decreased the lag time; however, LDL concentration had no effect. LDL‐induced Cu2+ reduction, however, was not affected by low initial O2 concentrations, suggesting that O2 is not required for LDL‐mediated reduction of Cu2+. Following the lag phase O2 consumption was dependent upon LDL or initial O2 concentrations; Cu2+ concentrations had little effect, suggesting that the propagation phase is more dependent on the presence of LDL lipids and O2 as substrates for the reaction. In summary, LDL peroxidation takes place in the presence of Cu2+ at low O2 tension; however, the reaction is dependent upon initial O2 concentrations; increases shorten the lag phase and accelerate O2 consumption.
Further characterization of rat dihydroceramide desaturase: Tissue distribution, subcellular localization, and substrate specificity
Authors: Causeret Catherine , Geeraert Luc , Van der Hoeven Gerd , Mannaerts Guy P. , Van Veldhoven Paul P.
Published: November 2006

The introduction of the double bond in the sphingoid backbone of sphingolipids occurs at the level of dihydroceramide via an NADPH‐dependent desaturase, as discovered in permeabilized rat hepatocytes. In the rat, the enzyme activity, which has now been further characterized, appeared to be mostly enriched in liver and Harderian gland. By means of subcellular fractionation of rat liver homogenates and density gradient separation of microsomal fractions, the desaturase was localized to the endoplasmic reticulum. Various detergents were inhibitory to the enzyme, and maximal activities were obtained in the presence of NADPH and when the substrate was complexed to albumin. In the presence of albumin, the chain length of the fatty acid of the truncated dihydroceramides hardly affected the activity. Finally, in view of a likely evolutionary relationship between desaturases and hydroxylases, the formation of hydroxylated intermediates was analyzed. No evidence for their presence was found under our assay conditions.
Discovery of an 11(R)‐ and 12(S)‐lipoxygenase activity in ovaries of the mussel Mytilus edulis
Authors: Coffa Gianguido , Hill Elizabeth M.
Published: November 2006

Eicosanoid biosynthesis was investigated in mussel gonads by incubation of tissue homogenates with radiolabeled arachidonic acid and analysis of the products by radio‐high‐performance liquid chromatography. No radiolabeled metabolites were formed in homogenates of testes, but two major metabolites were synthesized by ovarian preparations. The radiolabeled metabolites were analyzed by mass spectrometry and chiral chromatography and identified as 11(R)‐hydroxy‐5,8,12,14‐eicosatetraenoic acid and 12(S)‐hydroxy‐5,8,10,14‐eicosatetraenoic acid. In addition, four other nonlabeled metabolites, formed from endogenous substrates, were detected in ovarian extracts. Their structures, determined by mass spectrometric analysis, were the corresponding 11‐ and 12‐hydroxy analogs formed from eicosapentaenoic acid (11‐HEPE and 12‐HEPE) and 9‐hydroxy‐6,10,12,15‐octadecatetraenoic acid (9‐HOTE) and 13‐hydroxy‐6,9,11,15‐octadecatetraenoic acid formed from stearidonic acid. The biosynthesis of the 11‐ and 12‐hydroxy products was calcium dependent, localized to the 100,000×g supernatant cell fraction, and was inhibited by nordihydroguaiaretic acid, but not inhibited by the prostaglandin synthase inhibitors aspirin and indomethacin, or the monoxygenase inhibitor proadifen. Together these data suggested that both the 11(R)‐ and 12(S)‐hydroxy products were formed from lipoxygenase‐type enzymes. Incubation of homogenates of immature ovaries with eicosapentaenoic acid revealed the major product to be 12‐HEPE, whereas in mature ovaries mainly 11‐HEPE was formed. Extraction of spawned eggs with methanol revealed that predominantly 11‐HEPE and 9‐HOTE were formed from endogenous substrates. This study shows that female gonads of the mussel express an 11(R)‐ and 12(S)‐lipoxygenase activity whose expression is dependent on differentiation of the ovary.
Evidence for the mitochondrial biosynthesis of 3R‐hydroxy‐5Z,8Z,11Z,14Z‐eicosatetraenoic acid in the yeast Dipodascopsis uninucleata
Authors: Fox Simon R. , Hamberg Mats , Friend John , Ratledge Colin
Published: November 2006

The biosynthesis of 3R‐hydroxy‐5Z,8Z,11Z,14Z‐eicosatetraenoic acid (3R‐HETE) from arachidonic acid (20∶4n−6) by the hyphal‐forming yeast, Dipodascopsis uninucleata, in cell‐free enzyme extracts required CoASH, ATP, NAD+ and Mg2+; 3R‐HETE was present as the CoA derivative in enzyme extracts and its biosynthesis was associated with mitochondria. Its synthesis was high from arachidonoyl‐CoA (15% conversion of the substrate; 22 nmol mg protein−1·h), but significantly higher from trans‐2‐arachidonoyl‐CoA (53 nmol mg protein−1·min). Aspirin, an inhibitor of prostaglandin endoperoxide synthase synthase (cyclooxygenase), did not significantly inhibit 3R‐HETE biosynthesis in enzyme extracts, as opposed to antimycin A (46% inhibition). The chirality of 3‐HETE was 95% R and 5% S. 3R‐HETE has the same chirality as the products of peroxisomal enoyl‐CoA hydratases of Neurospora crassa and Saccharomyces cerevisiae; the difference appears to be that in D. uninucleata the R‐enantiomers are synthesized in mitochondria. Exogenously supplied eicosapentaenoic acid was converted to 3‐hydroxy 5Z,11Z,14Z,17Z‐eicosapentaenoic acid by cell‐free enzyme extracts though there was no requirement for a 5Z,8Z‐diene structure for the biosynthesis of 3‐hydroxylated fatty acids as 3‐hydroxy‐8Z,11Z,14Z, and 3‐hydroxy‐11Z,14Z,17Z‐eicosatrienoic acids were synthesized from the corresponding fatty acids. We found no evidence for the synthesis of the prostaglandins F2α and F2.
Human, but not bovine, oxidized cerebral spinal fluid lipoproteins disrupt neuronal microtubules
Authors: Neely M. D. , Swift L. L. , Montine T. J.
Published: November 2006

Cerebral spinal fluid (CSF) lipoproteins have become a focus of research since the observation that inheritance of particular alleles of the apolipoprotein E gene affects the risk of Alzheimer's disease (AD). There is evidence of increased lipid peroxidation in CSF lipoproteins from patients with AD, but the biological significance of this observation is not known. A characteristic of the AD brain is a disturbance of the neuronal microtubule organization. We have shown previously that 4‐hydroxy‐2(E)‐nonenal, a major product of lipid peroxidation, causes disruption of neuronal microtubules and therefore tested whether oxidized CSF lipoproteins had the same effect. We exposed Neuro 2A cells to human CSF lipoproteins and analyzed the microtubule organization by immunofluorescence. In vitro oxidized human CSF lipoproteins caused disruption of the microtubule network, while their native (nonoxidized) counterparts did not. Microtubule disruption was observed after short exposures (1 h) and lipoprotein concentrations were present in CSF (20 μg/mL), conditions that did not result in loss of cell viability. Importantly, adult bovine CSF lipoproteins, oxidized under identical conditions, had no effect on the microtubule organization of Neuro 2A cells. Comparison of human and bovine CSF lipoproteins revealed similar oxidation‐induced modifications of apolipoproteins E and A‐I as analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and Western blotting. Fatty acid analysis revealed substantially lower amounts of unsaturated fatty acids in bovine CSF lipoproteins, when compared to their human counterparts. Our data therefore indicate that oxidized human CSF lipoproteins are detrimental to neuronal microtubules. This effect is species‐specific, since equally oxidized bovine CSF lipoproteins left the neuronal microtubule organization unchanged.
Involvement of the ceramide signaling pathway in modulating the differentiated state of porcine thyroid cells
Authors: Schneider C. , Delorme N. , Buisson‐Legendre N. , Bellon G. , Emonard H. , El Btaouri H. , Hornebeck W. , Haye B. , Martiny L.
Published: November 2006

Neutral sphingomyelinase (Smase) is a cell membrane‐associated phospholipase that hydrolyzes sphingomyelin to phosphocholine and ceramide, a lipid second messenger involved in cell differentiation and/or apoptosis. We first evidenced that porcine cultured thyroid cells could express neutral Smase activity even if thyrotropin (TSH), an essential hormone in thyroid cell differentiation, was found to induce a 1.7‐fold decrease in Smase activity. Triggering the ceramide pathway by exogenous addition of neutral bacterial Smase (0.1 U/mL for 48 h), which transiently increased ceramide level by fourfold, drastically modified thyroid cell morphology. The follicle‐like structures generated by TSH were disrupted, and the Smase‐induced cell spreading was accompanied by a parallel loss of cell ability to iodinate proteins as well as a decrease of the adenylate cyclase system response. These inhibitory effects have been reproduced using short‐chain exogenous ceramide analogs (C2‐ceramides). Overall these data showed that ceramides emerged as potential mediators of dedifferentiation in thyroid cells.
Modulation of arachidonate and docosahexaenoate in Morone chrysops larval tissues and the effect on growth and survival
Authors: Harel Moti , Lund Eric , Gavasso Sonja , Herbert Ryan , Place Allen R.
Published: November 2006

The extent to which extreme dietary levels of arachidonate (AA) and/pr docosahexaenoate (DHA) modulate lipid composition in the body tissues and consequently affect growth and survival in freshwater Morone larvae species was examined. White bass, M. chrysops, larvae (day 24–46) were fed Artemia nauplii enriched with algal oils containing varying proportions of AA and DHA (from 0 to over 20% the total fatty acids). Growth was significantly reduced (P<0.05) in larvae fed a DHA‐deficient Artemia diet. Increases in dietary levels of AA also were associated with a significant growth reduction. However, the inhibitory effect of AA on larvae growth could be suppressed by the dietary addition of DHA (at a level of 21.6% of the total fatty acids in enrichment lipids). Larval brain+eyes tissue accumulated over 10 times more DHA than AA in its structural lipids (phosphatidylcholine, phosphatidylethanolamine) at any dietary ratio. In contrast, DHA accumulation, as compared to AA, in gill lipids declined considerably at higher than 10∶1 DHA/AA tissue ratios. DHA and eicosapentaenoic acid (EPA) contents in brain+eyes tissue were most sensitive to competition from dietary AA, being displaced from the tissue at rates of 0.36±0.07 mg DHA and 0.46±0.11 mg EPA per mg increase in tissue AA, and 0.55±0.14 mg AA per mg increase in tissue DHA. On the other hand, AA and EPA levels in gill tissue were most sensitive to dietary changes in DHA levels; AA was displaced at rates of 0.37±0.11 mg, whereas EPA increased at rates of 0.68±0.28 mg per mg increase in tissue DHA. Results suggest that balanced dietary DHA/AA ratios (that allow DHA/AA ratios of 2.5∶1 in brain+eyes tissue) promote a high larval growth rate, which also correlates with maximal regulatory response in tissue essential fatty acids.
Effects of fructose and troglitazone on phospholipid fatty acid composition in rat skeletal muscle
Authors: Clore John N. , Li Jing , Rizzo William B.
Published: November 2006

Skeletal muscle phospholipid fatty acid (PLFA) composition is associated with insulin sensitivity in animal models and in man. However, it is not clear whether changes in insulin sensitivity cause a change in PLFA composition or vice versa. The present studies have examined the effects of agents known to increase or decrease insulin sensitivity on PLFA composition of the major phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), in soleus and extensor digitorum longus muscle. Four groups of Sprague‐Dawley rats— control, 0.2% troglitazone (Tgz), 60% fructose fed, and fructose + Tgz—were treated for 3 wk. Fructose feeding was associated with a decrease in muscle membrane polyunsaturated fatty acids (PUFA) and n‐3 fatty acids in both PC and PE. Administration of Igz alone resulted in an increase in liver (3.75±0.93 to 6.93±1.00 μmol/min/mg tissue, P<0.05) and soleus muscle (0.34±0.03 to 0.67±0.09 μmol/min/mg, P<0.01) elongase activity, which would be expected to increase membrane PUFA. However, Tgz decreased PLFA associated with greater insulin sensitivity (e.g., PUFA and n‐3 fatty acids) and increased PLFA associated with decreased insulin sensitivity (16∶0 and n‐6 fatty acids) in both PC and PE. Co‐administration of fructose and Tgz did not reverse the decrease in PUFA observed with fructose alone. We conclude that the improvement in insulin sensitivity reported with Tgz is associated with an apparently paradoxical effect to decrease PUFA and n‐3 PLFA composition in rat skeletal muscle. These studies suggest that Tgz‐mediated increases in insulin sensitivity do not result in improved PLFA composition.
Phospholipid identification and quantification of membrane vesicle subfractions by31P−1H two‐dimensional nuclear magnetic resonance
Authors: Larijani B. , Poccia D. L. , Dickinson L. Charles
Published: November 2006

An approach to the direct quantification of phospholipids from two‐dimensional31P−1H nuclear magnetic resonance (NMR) spectroscopy with isotropic proton mixing has been developed as a general method for phospholipid analysis of minor membrane vesicle subfractions. Membrane vesicles were subfractionated by sedimentation to density equilibrium in a sucrose gradient, and a modified Folch method was employed to extract their phospholipids. The coefficient for the NMR detection efficiency of each phospholipid and the relative mole percentage of the phospholipids present in the membrane vesicles were calculated. We demonstrate low detection limits such that relative concentrations of phospholipids in membrane subfractions may be determined even in the submicromolar range.
Linoleic acid metabolism in the spontaneously diabetic rat: Δ6‐desaturase activity vs. product/precursor ratios
Authors: Brown J. E. , Lindsay R. M. , Riemersma R. A.
Published: November 2006

The activity of Δ6‐desaturase of linoleic acid, a rate‐limiting step in the formation of arachidonic acid, is decreased in animal models of severe, uncontrolled diabetes. The aim of the study was to measure the activity of liver microsomal Δ6‐desaturase of spontaneously diabetic BioBreeding/Edinburgh rats receiving subcutaneous insulin daily and of genetically related nondiabetic animals. The activity of Δ6‐desaturase was then compared with indices of activity (plasma lipid fatty acid product/precursor ratios) frequently used in human studies. Diabetic rats treated with insulin had 75±8% of the activity of microsomal Δ6‐desaturase of nondiabetic controls (P<0.05). Insulin withdrawal tended to reduce the activity further (61% of control), although the activity did not differ from insulin‐treated diabetic rats. The ratio of plasma phospholipid or cholesteryl ester γ‐linolenic over linoleic acid was not decreased in insulin‐treated diabetic rats. By contrast, the ratio of γ‐linolenic over linoleic acid of microsomes was almost three‐fold higher in insulin‐treated diabetic rats (P<0.05). The γ‐linolenic over linoleic acid ratio as an index of activity gave inconsistent results in insulin‐deprived rats. The ratio of γ‐linolenic over linoleic acid of cholesteryl esters did not differ between control and diabetic rats, nor did it correlate with microsomal Δ6‐desaturase activity. Furthermore, the index of Δ6‐desaturase activity, derived from the fatty acid composition of microsomal phospholipids, did not correlate with microsomal Δ6‐desaturase activity. Diabetes, even when controlled by regular insulin injections, reduces the metabolism of linoleic acid, but the effect is less than previously published. The fatty acid compositions of plasma and liver microsomal lipids are not reliable indices of Δ6‐desaturase activity in diabetes.
Purification, characterization, and molecular cloning of group I phospholipases A2 from the gills of the red sea bream, Pagrus major
Authors: Iijima Noriaki , Uchiyama Satoshi , Fujikawa Yukichi , Esaka Muneharu
Published: November 2006

Phospholipase A2 (PLA2) activity was investigated in various tissues of male and female red sea bream. In both male and female fishes, the specific activity of PLA2 in the gills was 70 times higher than that in other tissues, such as the adipose tissue, intestine, and hepatopancreas. Therefore, we tried to purify PLA2 from the gill filaments of red sea bream to near homogeneity by sequential chromatography on Q‐Sepharose Fast Flow, Butyl‐Cellulofine, and DEAE‐Sepharose Fast Flow columns, and by reversed‐phase high‐performance liquid chromatography. Two minor and one major PLA2, tentatively named G‐1, G‐2 and G‐3 PLA2, were purified, and all showed a single band with an apparent molecular mass of approximately 15 kDa by sodium dodecylsulfate‐polyacrylamide gel electrophoresis. The exact molecular mass values of G‐1, G‐2, and G‐3 PLA2 were 14,040, 14,040 and 14,005 Da, respectively. G‐1, G‐2, and G‐3 PLA2 had a Cys 11 and were all identical in N‐terminal amino acid sequences from Ala‐1 to Glu‐56. A full‐length cDNA encoding G‐3 PLA2 was cloned by reverse transcriptase‐polymerase chain reaction and rapid amplification of cDNA ends methods, and G‐3 PLA2 was found to be classified to group IB PLA2 from the deduced amino acid sequence. G‐1, G‐2, and G‐3 PLA2 had a pH optimum in an alkaline region at around pH 9–10 and required Ca2+ essentially for enzyme activity, using a mixed‐micellar phosphatidylcholine substrate with sodium cholate. These results demonstrate that three group 1 PLA2, G‐1, G‐2, and G‐3 PLA2, are expressed in the gill filaments of red sea bream.
Electron microscopy may reveal structure of docosahexaenoic acid‐rich oil withinSchizochytriumsp
Authors: Ashford A. , Barclay W. R. , Weaver C. A. , Giddings T. H. , Zeller S.
Published: November 2006

Schizochytriumsp. is an algae‐like microorganism utilized for commercial production of docosahexaenoic acid (DHA)‐rich oil and dried microalgae for use as a source of DHA in foods, feeds, and nutritional supplements. Electron microscopic analysis of whole cells ofSchizochytriumsp. employing sample preparation by high‐pressure freeze substitution suggests the presence of secondary and tertiary semicrystalline structures of triacylglycerols within the oil bodies inSchizochytriumsp. A fine secondary structure consisting of alternating light‐and darkstaining bands was observed inside the oil bodies. Dark bands were 29±1 Å in width, and light bands were 22±1 Å in width. The tertiary (three‐dimensional) structure may be a multilayered ribbon‐like structure which appears coiled and interlaced within the oil body. In freeze‐fracture photomicrographs,Schizochytriumoil bodies exhibited fracture planes with terraces averaging 52±7 Å in height which could correspond to the combined width of two halves of two light bands and one dark band observed in the high‐pressure freeze substitution photomicrographs. The results suggest that triacyglycerols withinSchizochytriumsp. oil bodies may be organized in a triple chainlength structure. High‐pressure freeze substitution electron micrographs of two other highly unsaturated oil‐producing species of microalgae,Thraustochytriumsp. andIsochrysis galbana, also revealed this fine structure, whereas microalgae containing a higher proportion of saturated oil did not. The results suggest that the staining pattern is not an artifact of preparation and that the triple chain‐length conformation of triacylglycerols inSchizochytriumsp. oil bodies may be caused by the unique fatty acid composition of the triacylglycerols.
Properties of a fluorescent bezafibrate derivative (DNS‐X). A new tool to study peroxisome proliferation and fatty acid β‐oxidation
Authors: Berlot Jean‐Pierre , Lutz Thomas , Malki Mutaspha Cherkaoui , Nicolas‐Frances Val rie , Jannin Brigitte , Latruffe Norbert
Published: November 2006

The first peroxisome proliferator‐activated receptor (PPAR) was cloned in 1990 by Issemann and Green. Many studies have reported the importance of this receptor in the control of gene expression of enzymes involved in lipid metabolic pathways including mitochondrial and peroxisomal fatty acid β‐oxidation, lipoprotein structure [apolipoprotein (apo) A2, apo Clll], and fatty acid synthase. By using radiolabeled molecules, it was shown that peroxisome proliferators bind and activate PPAR. As an alternative method, we developed a fluorescent dansyl (1‐dimethyl‐aminonaphthalene‐5‐sulfonyl) derivative peroxisome proliferator from bezafibrate (DNS‐X), a hypolipidemic agent that exhibits an in vitro peroxisome proliferative activity on rat Fao‐hepatic derived cultured cells. However, until now, the effect of this new compound on the liver of animals and subcellular localization was unknown. In addition to in vivo rat studies, we present a more efficient large‐scale technique of DNS‐X purification. Treating rats (DNS‐X in the diet at 0.3% w/w) for 6 d leads to a hepatomegaly and a marked increase in liver peroxisomal palmitoyl‐CoA oxidase activity. We also developed a method to localize and quantify DNS‐X in tissues or cell compartment organelles. The primarily cytosolic distribution of DNS‐X was confirmed by direct visualization using fluorescence microscopy of cultured Fao cells. Finally, transfection assay demonstrated that DNS‐X enhanced the PPARα activity as well as other peroxisome proliferators do.
Protective effect of phytic acid hydrolysis products on iron‐induced lipid peroxidation of liposomal membranes
Authors: Miyamoto Sayuri , Kuwata Goro , Imai Masatake , Nagao Akihiko , Terao Junji
Published: November 2006

Beneficial effects of dietary phytic acid (myo‐inositol hexaphosphate; IP6) have often been explained by its strong iron ion‐chelating ability, which possibly suppresses iron ion‐induced oxidative damage in the gastrointestinal tract. Because phytic acid is hydrolyzed during digestion, this work aimed to know whether its hydrolysis products (IP2′ IP3′, IP4′ and IP5) could still prevent iron ion‐induced lipid peroxidation. Studies using liposomal membranes demonstrated that hydrolysis products containing three or more phosphate groups are able to inhibit iron ion‐induced lipid peroxidation although their effectiveness decreased with dephosphorylation. Similarly, they also prevented iron ion‐induced decomposition of phosphatidylcholine hydroperoxide. These results demonstrate that intermediate products of phytic acid hydrolysis still possess iron ion‐chelating ability, and thus they can probably prevent iron ion‐induced lipid peroxidation in biological systems.
New cyclopentenone fatty acids formed from linoleic and linolenic acids in potato
Authors: Hamberg Mats
Published: November 2006

[1‐14C]Linoleic acid was incubated with a whole homogenate preparation from potato stolons. The reaction product contained four major labeled compounds, i.e., the α‐ketol 9‐hydroxy‐10‐oxo‐12(Z)‐octadecenoic acid (59%), the epoxy alcohol 10(S),11(S)‐epoxy‐9(S)‐hydroxy‐12(Z)‐octadecenoic acid (19%), the divinyl ether colneleic acid (3%), and a new cyclopentenone (13%). The structure of the last‐mentioned compound was determined by chemical and spectral methods to be 2‐oxo‐5‐pentyl‐3‐cyclopentene‐1‐octanoic acid (trivial name, 10‐oxo‐11‐phytoenoic acid). Steric analysis demonstrated that the relative configuration of the two side chains attached to the five‐membered ring was cis, and that the compound was a racemate comprising equal parts of the 9(R), 13(R) and 9(S), 13(S) enantiomers. Experiments in which specific trapping products of the two intermediates 9(S)‐hydroperoxy‐10(E), 12(Z)‐octadecadienoic acid and 9(S), 10‐epoxy‐10, 12(Z)‐octadecadienoic acid were isolated and characterized demonstrated the presence of 9‐lipoxygenase and allene oxide synthase activities in the tissue preparation used. The allene oxide generated from linoleic acid by action of these enzymes was further converted into the cyclopentenone and α‐ketol products by cyclization and hydrolysis, respectively. Incubation of [1‐14C]linolenic acid with the preparation of potato stolons afforded 2‐oxo‐5‐[2′(Z)‐pentenyl]‐3‐cyclopentene‐1‐octanoic acid (trivial name, 10‐oxo‐11, 15(Z)‐phytodienoic acid), i.e., an isomer of the jasmonate precursor 12‐oxo‐10, 15(Z)‐phytodienoic acid. Quantitative determination of 10‐oxo‐11‐phytoenoic acid in linoleic acid‐supplied homogenates of different parts of the potato plant showed high levels in roots and stolons, lower levels in developing tubers, and no detectable levels in leaves.
Biased distribution of the branched‐chain fatty acids in ceramides of vernix caseosa
Authors: Oku Hirosuke , Mimura Kunio , Tokitsu Yumi , Onaga Kyoko , Iwasaki Hironori , Chinen Isao
Published: November 2006

The compositions of ester‐ and amide‐linked fatty acids from ceramides of human vernix caseosa were described with emphasis on the distribution of the branched‐chain fatty acid (BCFA). Two novel ceramides were isolated from vernix caseosa in the course of this study: the acylated type of esterified α‐OH‐hydroxyacid/sphingosine ceramide (Cer[EAS]) and nonacylated type of non‐OH fatty acid/hydroxysphingosine ceramide (Cer[NH]). Their chemical structures were identified by nuclear magnetic resonance and chemical procedure. The Cer[EAS] was an acylceramide and consisted of the highest concentrations of ester‐ and amide‐linked BCFA (62 and 67%, respectively). The iso‐ or anteiso‐branching structures of the aliphatic chains were confirmed by the mass spectra of their picolinyl or pyrrolidide derivatives. As a whole, amide‐linked fatty acids of ceramides 1–7 and Cer[NH] were normal types of straight‐chain fatty acids with or without α‐ or ω‐hydroxylation. The BCFA concentrations of amide‐linked fatty acids in these ceramides (ceramides 1–7 and Cer[NH]) were low and less than 10%. The BCFA thus occurred exclusively in a novel acylceramide of Cer[EAS] in the vernix caseosa.
High dietary 18∶3n−3 increases the 18∶3n−3 but not the 22∶6n−3 content in the whole body, brain, skin, epididymal fat pads, and muscles of suckling rat pups
Authors: Bowen Raffick A. R. , Clandinin Michael T.
Published: November 2006

The objective of this study was to test the hypothesis that increasing maternal dietary 18∶3n−3 by decreasing the 18∶2n−6/18∶3n−3 ratio will increase the 18∶3n−3 and 22∶6n−3 content of the whole body, liver, skin (epidermis, dermis, and subcutaneous tissue), epididymal fat pads, and muscles (arms and legs) of 2‐wk‐old rat pups. Sprague‐Dawley dams at parturition were fed semipurified diets containing either a low (18∶2n−6 to 18∶3n−3 ratio of 24.7∶1) or a high (18∶2n−6 to 18∶3n−3 ratio of 1.0∶1) 18∶3n−3 fatty acid content. During the first 2 wk of life, rat pups received only their dams' milk. Fatty acid composition of the pups' stomach contents (dams' milk), whole body, brain, liver, skin, epididymal fat pads, and muscles was determined. The stomach fatty acid composition of 18∶3n−3 reflected the dams' diet. The content of 18∶3n−3 in whole body, brain, liver, skin, epididymal fat pads, and muscles was significantly (P<0.05) greater in rat pups fed the high compared with the low 18∶3n−3 fatty acid diet. The 22∶6n−3 content of the whole body, brain, skin, epididymal fat pads, and muscles was not quantitatively different in rat pups fed either the low or high 18∶3n−3 fatty acid diet. The 20∶5n−3 and 22∶5n−3 content of the whole body, skin, and epididymal fat pads was significantly increased in rat pups fed the high compared with the low 18∶3n−3 fatty acid diet. High content of 18∶3n−3 was found in the skin of rat pups fed either a low or high 18∶3n−3 fatty acid diet. These findings demonstrate that high maternal dietary 18∶3n−3 significantly increases the 18∶3n−3 but not the 22∶6n−3 content of the whole body, brain, skin, epididymal fat pads, and muscles with approximately 39 and 41% of the whole body 18∶3n−3 content being deposited in the skin of suckling rat pups fed either the low or high 18∶3n−3 diet, respectively.
Influence of sources of dietary oils on the life span of stroke‐prone spontaneously hypertensive rats
Authors: Ratnayake W. M. N. , Plouffe L. , Hollywood R. , L'Abbé M. R. , Hidiroglou N. , Sarwar G. , Mueller R.
Published: November 2006

In recent studies, the life span of stroke‐prone spontaneously hypertensive (SHRSP) rats was altered by a variety of dietary fats. It was relatively shorter in rats fed canola oil as the sole source of fat. The present study was performed to find out whether the fatty acid profile and the high content of sulfur compounds in canola oil could modulate the life span of SHRSP rats. SHRSP rats (47 d old, n=23/group) were matched by body weight and systolic blood pressure and fed semipurified diets containing 10% canola oil, high‐palmitic canola oil, low‐sulfur canola oil, soybean oil, high‐oleic safflower oil, a fat blend that mimicked the fatty acid composition of canola oil, or a fat blend high in saturated fatty acids. A 1% sodium chloride solution was used as drinking water to induce hypertension. After consuming the diets for 37 d, five rats from each dietary group were killed for collection of blood and tissue samples for biochemical analysis. The 18 remaining animals from each group were used for determining their life span. The mean survival time of SHRSP rats fed canola oil (87.4±4.0 d) was not significantly different (P>0.05) from those fed low‐sulfur canola oil (89.7±8.5 d), suggesting that content of sulfur in canola oil has no effect on the life span of SHRSP rats. The SHRSP rats fed the noncanola oil‐based diets lived longer (mean survival time difference was 6–13 d, P<0.05) than those fed canola and low‐sulfur canola oils. No marked differences in the survival times were observed among the noncanola oil‐based groups. The fatty acid composition of the dietary oils and of red blood cells and liver of SHRSP rats killed after 37 d of treatment showed no relationship with the survival times. These results suggest that the fatty acid profile of vegetable oils plays no important role on the life span of SHRSP rat. However, phytosterols in the dietary oils and in liver and brain were inversely correlated with the mean survival times, indicating that the differential effects of vegetable oils might be ascribed, at least partly, to their different phytosterol contents.
Dietary fat‐induced suppression of lipogenic enzymes in B/B rats during the development of diabetes
Authors: Cheema S. K. , Clandinin M. Thomas
Published: November 2006

This study was designed to determine the level of inhibition of gene transcription by the reduction in insulin levels upon the onset of diabetes in spontaneously diabetic B/B rats and if reducing the level of polyunsaturated fatty acids (PUFA) in the diet will increase lipogenic enzyme activity. Control (eight animals per group) and spontaneously diabetic B/B male weanling rats (25 animals per group) were fed semipurified diets containing 20% (w/w) fat of either low (0.25) or high (1.0) polyunsaturated to saturated (P/S) fatty acid ratio. Rats were killed at the onset of diabetes [blood glucose level of ≅100 mg/dL (5.55 mM)] and as they became highly diabetic [blood glucose level of ≅400 mg/dL (22.22 mM)]. Total RNA was extracted from liver, and the relative amount of mRNA coding for fatty acid synthase (FAS), acetyl‐CoA carboxylase, malic enzyme, pyruvate kinase, and phosphoenolpyruvate carboxykinase was determined. Liver enzyme activities were also measured. The mRNA levels for FAS, acetyl‐CoA carboxylase, and malic enzyme decreased compared to control animals. The mRNA level for pyruvate kinase decreased at the onset of diabetes as compared to control animals. Feeding animals the low P/S diet treatment elevated the level of mRNA and lipogenic enzyme activity compared to animals fed the high P/S diet treatment, suggesting that the effect of PUFA on lipogenic enzymes is through a direct effect on gene expression.
Lipid composition of erythrocytes and thrombocytes of a subantarctic seabird, the king penguin
Authors: Fayolle Christine , Leray Claude , Ohlmann Philippe , Gutbier Geneviève , Cazenave Jean‐Pierre , Gachet Christian , Groscolas René
Published: November 2006

Phospholipid (PL) compositions and fatty acid (FA) patterns of PL were determined in the erythrocytes and blood thrombocytes of a seabird, the king penguin, living in the subantarctic area and feeding on prey rich in n−3 polyunsaturated FA. Results were compared between birds in three different physiological states (breeding and molting adults, chicks) to those reported for other birds. In erythrocytes, the ratios of cholesterol to PL and of sphingomyelin to phosphatidylcholine (PC) were lower than in other birds. The PL distribution was similar to those previously reported in the hen and pigeon. In contrast to other birds, cardiolipin levels were unexpectedly high (4%). Very long chain n−3 FA were abundant (13–27%) in phosphatidylethanolamine (PE), phosphatidylserine and PC, probably in relation to the natural diet of these birds. Among n−3 FA, 22∶6n−3 was the most abundant in all PL (2−20%), whereas the highest levels of arachidonic acid were observed in PE (14%). In thrombocytes, the PL distribution and FA composition of the main PL (PC, PE) differed from those of erythrocytes, and in particular, levels of n−3 FA (9–12%) were 1.5–2 times lower. The highest levels of arachidonic acid were found in phosphatidylinositol (24%). The lipid profile of penguin erythrocytes could contribute to the efficiency of blood circulation and oxygen delivery in microvascular beds, thus favoring diving capacity of these animals. Our observations do not support the hypothesis of a common origin of avian thrombocytes and erythrocytes.
Metabolism of 1‐acyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoethanolamine in castor oil biosynthesis
Authors: Lin Jiann‐Tsyh , Lew Karen M. , Chen Jennifer M. , Iwasaki Yugo , McKeon Thomas A.
Published: November 2006

We have examined the role of 2‐oleoyl‐PE (phosphatidylethanolamine) in the biosynthesis of triacylglycerols (TAG) by castor microsomes. In castor microsomal incubation, the label from 14C‐oleate of 1‐palmitoyl‐2‐[1‐14C]oleoyl‐sn‐glycero‐3‐phosphoethanolamine is incorporated into TAG containing ricinoleate. The enzyme characteristics, such as optimal pH, and the effect of incubation components of the oleoyl‐12‐hydroxylase using 2‐oleoyl‐PE as incubation substrate are similar to those for 2‐oleoyl‐PC (phosphatidylcholine). However, compared to 2‐oleoyl‐PC, 2‐oleoyl‐PE is a less efficient incubation substrate of oleoyl‐12‐hydroxylase in castor microsomes. Unlike 2‐oleoyl‐PC, 2‐oleoyl‐PE is not hydroxylated to 2‐ricinoleoyl‐PE by oleoyl‐12‐hydroxylase and is not desaturated to 2‐linoleoyl‐PE by oleoyl‐12‐desaturase. We have demonstrated the conversion of 2‐oleoyl‐PE to 2‐oleoyl‐PC and vice versa. The incorporation of label from 2‐[14C]oleoyl‐PE into TAG occurs after its conversion to 2‐oleoyl‐PC, which can then be hydroxylated or desaturated. We detected neither PE‐N‐monomethyl nor PE‐N,N‐dimethyl, the intermediates from PE to PC by N‐methylation. The conversion of 2‐oleoyl‐PE to 2‐oleoyl‐PC likely occurs via hydrolysis to 1,2‐diacyl‐sn‐glycerol by phospholipase C and then by cholinephosphotransferase. This conversion does not appear to play a key role in driving ricinoleate into TAG.