bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of inositol sphingophospholipids from Phytophthora parasitica Dastur
Authors: Bruneteau Maud , Fournol Florence , Gandon Corinne , Becchi Michel , Pivot Véronique
Published: November 2006

Several inositol sphingophospholipids (ISPL) were isolated from mycelia of Phytophthora parasitica Dastur, a phytopathogenic fungus of carnation. The ISPL structures were determined by fast atom bombardment. All ISPL consisted of ceramides linked to inositol phosphate. We investigated the effect of growth conditions on the ISPL produced in four different media that are commonly used for fungal cultures. We showed that P. parasitica Dastur synthesized four major classes of compounds with molecular weights of Mr=751, 807, 835, and 849 containing the 16:1 base and the 16:0 or 20:0 or 22:0 or 22h:1 N‐acyl group. The relative abundance of the different ISPL is dependent on growth conditions.
Nuclear magnetic resonance characterization of 6α‐chloro‐5β‐cholestane‐3β,5‐diol formed from the reaction of hypochlorous acid with cholesterol
Authors: Carr Anitra C. , Winterbourn Christine C. , Blunt John W. , Phillips Andrew J. , Abell Andrew D.
Published: November 2006

Hyopochlorous acid generated by neutrophil myeloperoxidase has been shown to convert cholesterol into three different chlorohydrin isomers which previously had not been fully characterized. We have reacted hypochlorous acid with cholesterol/1,2‐dipalmitoyl phosphatidylcholine liposomes to give these three major products and established that they are 6β‐chloro‐5α‐cholestane‐3β,5‐diol (chlorohydrin 1), 5α‐chloro‐6β‐cholestane‐3β,6‐diol (chlorohydrin 2) and 6α‐chloro‐5β‐cholestane‐3β,5‐diol (chlorohydrin 3). These products were separated by thin‐layer chromatography and fully characterized by 1H, 13C, attached proton test, doublequantum correlation spectroscopy, total correlation spectroscopy, heteronuclear multiple bond correlation and heteronuclear multiple quantum coherence nuclear magnetic resonance spectroscopy.
Separation and quantitation of linoleic acid oxidation products in mammary gland tissue from mice fed low‐ and high‐fat diets
Authors: Johnson Jennifer A. , Blackburn Mary L. , Bull Arthur W. , Welsch Clifford W. , Throck Watson J.
Published: November 2006

We have developed an assay for the isolation and quantitation by gas chromatography‐mass spectrometry (GC‐MS) of free 9‐ and 13‐hydroxyoctadecadienoic acid (9‐HODE, 13‐HODE) in the mammary glands of female mice. Internal standards consisting of 18O2‐labeled analogs of 9‐ and 13‐HODE are added to pulverized frozen tissue prior to extraction with ethanol. Nonlipid materials are removed in a chloroform/methanol/water extraction step. The remaining lipid material is methylated with ethereal diazomethane, and much of the nonoxygenated fatty acid methyl esters are removed via silica solid‐phase extraction. Samples are either further derivatized with bis(trimethylsilyl)trifluoroacetamide to form the trimethylsilyl ethers for quantitative analysis by GC‐MS or are analyzed as the methyl esters by chiral high‐performance liquid chromatography to determine the enantiomeric distribution of the 9‐ and 13‐HODE. The extraction and quantitation protocol was applied to the analysis of mammary glands for free 9‐ and 13‐HODE from mice fed isocaloric diets containing 20% corn oil, 5% corn oil, or 20% beef tallow. Chiral analysis of the products showed higher production of 13(S)‐HODE relative to 13(R)‐HODE; the enantiomeric excess is most likely due to enzymatic production of 13‐HODE superimposed on a background of autoxidative production of 13(R)‐plus 9(S)‐ and 9(R)‐HODE. In addition, the effect of sample handling and storage conditions on the formation of 9‐ and 13‐HODE in the samples was assessed by exposing aliquots of a common pool of rat mammary gland tissue to specified conditions prior to analysis. This methodology will be important during investigations of the contribution of linoleate oxidation products to the enhancement of mammary tumorigenesis by dietary fat.
Effect of liposome‐encapsulated hemoglobin on triglyceride, total cholesterol, low‐density lipoprotein, and high‐density lipoprotein cholesterol measurements
Authors: Abdullah F. , Whiteford M. , Mathiak G. , Ovadia P. , Rudolph A. , Neville L. F. , Rabinovici R.
Published: November 2006

The present study investigated the effect of liposome‐encapsulated hemoglobin (LEH), an experimental oxygen‐carrying resuscitation fluid, on triglyceride, total cholesterol, and low density lipoprotein (LDL), and high density lipoprotein (HDL) cholesterol measurements. In vivo, the intravenous infusion of LEH (5.6 mL/kg, n=6) elevated serum triglycerides (+92% vs. baseline, P<.05), total cholesterol (+25% vs. baseline, P<.01), LDL cholesterol (+72% vs. baseline, P<.01) and had no effect on serum HDL cholesterol. In addition, LEH did not alter the elevation in serum triglycerides (+302% vs. baseline, P<.01) and LDL cholesterol (+86% vs. baseline, P<.01) induced by lipopolysaccharide (3.6 mg/kg, i.v., n=6). Ex vivo, measurements of triglycerides and total cholesterol as well as LDL and HDL cholesterol in whole blood from naive rats were not changed by the addition of LEH (0–50%, n=6). In vitro, the addition of a fixed concentration of LEH (50%, n=6) to varying concentrations of cholesterol solution (0–50%), or vice versa, had no effect on cholesterol determination. It is therefore concluded that LEH only minimally affects serum levels of triglycerides, total cholesterol, LDL cholesterol, and HDL cholesterol and does not interfere with their measurement.
The effect of dietary arachidonic acid on platelet function, platelet fatty acid composition, and blood coagulation in humans
Authors: Nelson G. J. , Schmidt P. C. , Bartolini G. , Kelley D. S. , Kyle David
Published: November 2006

Arachidonic acid (AA) is the precursor of thromboxane and prostacyclin, two of the most active compounds related to platelet function. The effect of dietary AA on platelet function in humans is not understood although a previous study suggested dietary AA might have adverse physiological consequences on platelet function. Here normal healthy male volunteers (n=10) were fed diets containing 1.7 g/d of AA for 50 d. The control diet contained 210 mg/d of AA. Platelet aggregation in the platelet‐rich plasma was determined using ADP, collagen, and AA. No statistical differences could be detected between the aggregation before and after consuming the high‐AA diet. The prothrombin time, partial thromboplastin time, and the antithrombin III levels in the subjects were determined also. There were no statistically significant differences in these three parameters when the values were compared before and after they consumed the high‐AA diet. The in vivo bleeding times also did not show a significant difference before and after the subjects consumed the high‐AA diet. Platelets exhibited only small changes in their AA content during the AA feeding period. The results from this study on blood clotting parameters and in vitro platelet aggregation suggest that adding 1.5 g/d of dietary AA for 50 d to a typical Western diet containing about 200 mg of AA produces no observable physiological changes in blood coagulation and thrombotic tendencies in healthy, adult males compared to the unsupplemented diet. Thus, moderate intakes of foods high in AA have few effects on blood coagulation, platelet function, or platelet fatty acid composition.
Increased dietary arachidonic acid enhances the synthesis of vasoactive eicosanoids in humans
Authors: Ferretti Aldo , Nelson Gary J. , Schmidt Perla C. , Kelley Darshan S. , Bartolini Giovanni , Flanagan Vincent P.
Published: November 2006

Data on the effect of dietary arachidonic acid (AA) (20∶4n‐6) on the synthesis of thromboxane and prostacyclin (PGI2) in humans are lacking. We measured the effect of 1.5 g/d (ca. 0.5 en%) of 20∶4n‐6 added isocalorically to a stabilization (low‐AA) diet on the excretion of 11‐dehydrothromboxane B2 (11‐DTXB2) and 2,3‐dinor‐6‐oxo‐PGF1α (PGI2‐M). In a crossover design, 10 healthy men, living in a metabolic unit, were fed a diet (low‐AA) containing 210 mg/d of 20∶4n‐6 for 65 d and an identical diet (high‐AA) that contained 1.5 g/d of additional 20∶4n‐6 for 50 d. Three‐day urine pools were collected at the end of each dietary period and analyzed for eicosanoids by gas chromatography‐electron capture negative ion‐tandem mass spectrometry. Mean excretion of 11‐dehydrothromboxane B2 was 515±76, 493±154, and 696±144 ng/d (SD; n=10) during the acclimation (15 d) low‐AA diet and high‐AA diet periods, respectively (41% increase from low‐AA to high‐AA diet, P=0.0037); mean excretion of PGI2‐M was 125±40, 151±36, and 192±55 ng/d (SD; n=10) during acclimation (15 d) low‐AA and high‐AA diets, respectively (27% increase from low‐AA to high‐AA diets; P=0.0143). Thus, both the metabolites of thromboxane and PGI2 increase on the high‐AA diet. Furthermore, both indicated changes in metabolite excretion may be associated with measurable effects on several physiologically significant cellular functions, such as platelet aggregation in vivo and inflammation in response to immune challenges.
Influence of dietary arachidonic acid on metabolism in vivo of 8 cis, 11 cis, 14‐eicosatrienoic acid in humans
Authors: Emken E. A. , Adlof R. O. , Duval S. M. , Nelson G. J.
Published: November 2006

This study investigated the influence of dietary arachidonic acid (20∶4n‐6) on Δ5 desaturation and incorporation of deuterium‐labeled 8cis, 11cis, 14‐eicosatrienoic acid (20∶3n‐6) into human plasma lipids. Adult male subjects (n=4) were fed diets containing either 1.7 g/d (H120∶4 diet) or 0.21 g/d (LO20∶4 diet) of arachidonic acid for 50 d and then dosed with a mixture containing ethyl esters of 20∶3n‐6[d4] and 18∶1n‐9[d2]. A series of blood samples was sequentially drawn over a 72‐h period, and methyl esters of plasma total lipid, triacylglycerol, phospholipids, and cholesteryl ester were analyzed by gas chromatography‐mass spectrometry. Based on the concentration of 20∶3n‐6[d4] in total plasma lipid, the estimated conversion of 20∶3n‐6[d4] to 20∶4n‐6[d4] was 17.7.±0.79% (HI20∶4 diet) and 2.13±1.44% (LO20∶4 diet). The concentrations of 20∶4n‐6[d4] in total plasma lipids from subjects fed the HI20∶4 and LO20∶4 diets were 2.10±0.6 and 0.29±0.2 μmole/mL plasma/mmole of 20∶3n‐6[d4] fed/kg of body weight. These data indicate that conversion of 20∶3n‐6[d4] to 20∶4n‐6[d4] was stimulated 7‐8‐fold by the HI20∶4 diet. Phospholipid acyltransferase was 2.5‐fold more selective for 20∶3n‐6[d4] than 18∶1n‐9[d2], and lecithin:cholesterol acyltransferase was 2‐fold more selective for 18∶1n‐9[d2] than 20∶3n‐6[d4]. These differences in selectivity were not significantly influenced by diet. Absorption of ethyl 20∶3n‐6[d4] was about 33% less than ethyl 18∶1n‐9[d2]. The sum of the n‐6 retroconversion products from 20∶3n‐6[d4] in total plasma lipids was about 2% of the total deuterated fatty acids. Neither absorption nor retroconversion appears to be influenced by diet.
Metal ion stimulation of phospholipase D‐like activity of isolated rat intestinal mitochondria
Authors: Madesh M. , Balasubramanian K. A.
Published: November 2006

Presence of phospholipase D‐like (PLD) activity in the intestinal mitochondria was identified using endogenous phospholipids as substrate. The enzyme had a pH optimum of 6.5, did not show trans‐phosphatidylation activity in the presence of ethanol or butanol, and the product formed was phosphatidic acid (PA). This was confirmed by separation of reaction products by high‐performance liquid chromatography and analysis of composition of the PA formed which gave phosphate/fatty acid ratio of 1∶2. PLD‐like activity was further confirmed by the formation of ethanolamine and choline as products of enzyme action. This activity was stimulated by various metal ions; when stimulated by Mg2+ and Ba2+, it hydrolyzed both phosphatidylcholine and phosphatidylethanolamine, and when stimulated by Ca2+, it preferentially hydrolyzed phosphatidylethanolamine. There was no requirement for sodium oleate for the PLD‐like activity in mitochondria. These results suggest that intestinal mitochondria have an active PLD‐like enzyme which differs in certain properties from phospholipase D from other tissues.
Membrane lipid metabolism and phospholipase activity in insect Spodoptera frugiperda 9 ovarian cells
Authors: Yeh Li‐Hong P. , Bajpai Rakesh K. , Sun Grace Y.
Published: November 2006

Although there is increasing use of insect ovarian Sf9 cells for the production of recombinant proteins, namely, via the baculovirus vector expression system, little is known about the lipids in the cell membrane and whether endogenous phospholipases are present for regulation of the cell membrane lipids. In this study, analysis of membrane lipids of Sf9 cells indicated the presence of phosphatidylethanolamine (PE) (diacyltype) and phosphatidylcholine as major phospholipids, followed by phosphatidylserine and phosphatidylinositol (PI), and only trace amounts of ethanolamine plasmalogen. These phospholipids contain high proportions of monoenoic fatty acids, e.g., 16∶1 and 18∶1, which comprise more than 70% of the total fatty acids although small amounts of polyunsaturated fatty acids such as 18∶2 and 20∶4 are also present. When Sf9 cells were incubated in a culture medium containing [14C]oleic acid and [14C]arachidonic acid, a large portion of the labels were incorporated into membrane phospholipids. Using [14C]arachidonoyl‐phospholipids as substrates for incubation with cell homogenate and subcellular fractions, results indicate the presence of a Ca2+‐independent phospholipase A (PLA2) in the Sf9 cell cytosol fraction. This PLA2 shows a high preference for hydrolysis of PE and is active at a pH range of 7–9. Unlike the brain cells which contain active phospholipase C (PLC) specific for phosphatidylinositol, only limited amount of diacylglycerol (DAG) was released from [14C]arachidonoyl‐PE in the Sf9 cells. Taken together, this study demonstrates active metabolism of membrane phospholipids in Sf9 cells, most likely mediated by acyltransferases and PLA2. Furthermore, despite the absence of PLC for PI, limited amount of DAG could be generated through hydrolysis of PE.
Effect of docosahexaenoic acid on mouse mitochondrial membrane properties
Authors: Stillwell William , Jenski Laura J. , Thomas Crump F. , Ehringer William
Published: November 2006

Long‐chain polyunsaturated (n‐3) fatty acids have been proposed to be involved in a wide variety of biological activities. In this study, mitochondrial docosahexaenoic acid (DHA) levels were increased by either dietary manipulation or by fusing the mitochondria with phospholipid vesicles made from 1‐stearoyl‐2‐docosahexaenoyl‐sn‐glycero‐3‐phosphocholine (18:0/22:6 PC). The fused mitochondria exhibited a DHA‐induced decrease in respiratory control index (RCI) and membrane potential and an increase in proton movement. The modified mitochondria also demonstrated an increase in fluidity (as detected by 1,6‐diphenyl‐1,3,5‐hexatriene anisotropy) and changes in membrane structure detected by the fluorescence probes MC540 and pyrene decanoate. Proton movement in lipid vesicles made from mitochondrial lipid extracts was shown to be enhanced by incorporated 18:0/22:6 PC. Mitochondria were isolated from young (5‐mon) and old (24‐mon) mice which were maintained on either a diet rich in saturated fats (hydrogenated coconut oil) or rich in n−3 polyunsaturated fats (menhaden oil). Mitochondrial bioenergetic function was followed by RCI, state 3 respiration, ATP level, and phosphate uptake. In addition, lipid composition, phospholipid area/molecule, and extent of lipid peroxidation were also determined. Decreases in RCI for the menhaden oil diet‐modified mitochondria paralleled those in which DHA levels were enhanced by fusion with phospholipid vesicles. RCI reductions are attributed to DHA‐induced increases in H+ movement, producing diminished mitochondrial membrane potentials. One purpose of this project was to determine if the deleterious effects of aging on mitochondrial bioenergetic function could be reversed by addition of n−3 fatty acids. The experiments reported here indicate that incorporation of long‐chain polyunsaturated n−3 fatty acids into mitochondrial membranes does not appear likely to reverse the effects of age on mitochondrial function.
The effect of dietary lipid on polyunsaturated fatty acid metabolism in Atlantic salmon (Salmo salar) undergoing parr‐smolt transformation
Authors: Bell J. Gordon , Tocher Douglas R. , Farndale Bruce M. , Cox David I. , McKinney Richard W. , Sargent John R.
Published: November 2006

The aim of this study was to measure the changes in lipid metabolism which occur during smoltification and seawater transfer in Atlantic salmon (Salmo salar). Duplicate groups of Atlantic salmon parr were fed diets containing either fish oil (FO) or a blend of linseed and rapeseed oils, vegetable oil (VO), from October (week 0) to seawater transfer in May (week 26). From May to August (weeks 26–43), all fish were fed a fish oil‐containing diet. Fatty acyl desaturation and elongation activity were followed in isolated hepatocytes incubated with radioactive 18:3n−3 and 18:2n−6. Metabolism of 18:3n−3 was consistently around 5‐fold greater than metabolism of 18:2n−6, and total metabolism of both substrate polyunsaturated fatty acids (PUFA) was increased in fish fed both VO and FO up to seawater transfer after which desaturation activities were reduced. Desaturation activities with both 18:3n−3 and 18:2n−6 were significantly greater in fish fed VO, compared to fish fed FO, at 22 and 26 wk. Arachidonic acid (20:4n−6; AA) in liver polar lipids (PL) of fish fed VO increased consistently from weeks 0–22 but varied after seawater transfer. In fish fed FO, AA in liver PL remained constant up to week 17 before increasing at seawater transfer and leveling off thereafter. Eicosapentaenoic acid (20:5n−3; EPA) in liver PL of fish fed VO decreased significantly from week 0–22 before rising at seawater transfer and increasing rapidly posttransfer. EPA in liver PL of fish fed FO showed a similar trend except EPA was always greater in the freshwater phase compared to fish fed VO. Docosahexaenoic acid (DHA) levels in liver PL of fish fed VO remained constant in the freshwater phase before increasing following seawater transfer. In fish fed FO, DHA in liver PL increased from weeks 0–17 reducing and leveling off postseawater transfer. The levels of PGF2α and PGF3α were measured in isolated gill cells stimulated with calcium ionophore A23187. PGF2α production in fish fed VO increased significantly between 0–7 wk before decreasing toward seawater transfer. After transfer, PGF2α production increased to a peak at 35 wk. PGF2α production in fish fed FO was not significantly altered during the trial period. The changes in PGF3α production were broadly similar to those occurring with PGF2α, but the latter was always in excess of the former (2‐to 4‐fold). Plasma chloride concentrations in fish subjected to seawater challenge at 20 wk were significantly lower in fish fed VO compared to those fed FO. This study has provided new information on the changes in lipid metabolism which accompany parr‐smolt transformation and suggests that diets which have a fatty acid composition more similar to that in aquatic invertebrates may be beneficial in effecting successful seawater adaptation.
Modifying the n−3 fatty acid content of the maternal diet to determine the requirements of the fetal and suckling rat
Authors: Guesnet Philippe , Alasnier Corinne , Alessandri Jean‐Marc , Durand Georges
Published: November 2006

During perinatal development, docosahexaenoic acid (22:6n−3) accumulates extensively in membrane phospholipids of the nervous system. To evaluate the n−3 fatty acid requirements of fetal and suckling rats, we investigated the accumulation of 22:6n−3 in the brain and liver of pup rats from birth to day 14 postpartum when their dams received increasing amounts of dietary 18:3n−3 (from 5 to 800 mg/100 g diet) during the pregnancy‐lactation period. The fatty acid composition of brain and liver phospholipids of pups, as well as that of dam's milk, was determined. At birth, brain 22:6n−3 increased regularly to reach the highest level when the maternal diet contained 800 mg 18:3n−3/100 g. On days 7 and 14 postpartum, brain 22:6n−3 plateaued at a maternal dietary supply of 200 mg/100 g. Docosapentaenoic acid (22:5n−6) had the opposite temporal pattern. The unusually high concentration of eicosapentaenoic acid (20:5n−3) in liver and dam's milk observed at the highest 18:3n−3 intake suggests an excessive dietary supply of this fatty acid. All these data suggest that the n−3 fatty acid requirements of the pregnant rat are around 400 mg 18:3n−3 and those of the lactating rat at 200 mg (i.e., 0.9 and 0.45% of dietary energy, respectively). The values for 18:3n−3 and 22:6n−3 milk content which allowed brain 22:6n−3 to reach a plateau value in suckling pups were 1% of total fatty acids and 0.9% (colostrum) to 0.2% (mature milk), respectively. These levels are similar to those recommended for infant formulas.
Nonionic surfactants. Regioselective synthesis of fatty acid esters of α‐ and β‐glucopyranose
Authors: De Luca Damiano , De Nino Antonio , Liguori Angelo , Procopio Antonio , Sindona Giovanni
Published: November 2006

Lipophilic esters of saccharides belong to the family of nonionic surfactants widely employed in pharmaceutical and cosmetics formulations. A very simple method is presented whereby 6‐O‐esters of α‐ and β‐glucose can be prepared and isolated. Good results have been obtained in the synthesis of 6‐O‐oleyl derivatives by simple acylation with appropriate oleyl chloride. The condensing agent bis(2‐oxo‐3‐oxazolidinyl) phosphinic chloride (BOP‐Cl) allows the preparations of the same esters with better regioselectivity but with yields strongly dependent on the aliphatic chain length of the carboxylic acids employed.
Novel fatty acid esters of (7E, 12E, 18R, 20Z)‐variabilin from the marine sponge Ircinia felix
Authors: Martínez Alejandro , Duque Carmenza , Fujimoto Yoshinori
Published: November 2006

The methanolic extract of the marine sponge Ircinia felix has yielded nine novel fatty acid esters, (7E, 12E, 18R, 20Z)‐variabilin (5Z, 9Z)‐22‐methyltricosadienoate, (7E, 12E, 18R, 20Z)‐variabilin (5Z, 9Z)‐tetracosadienoate, (7E, 12E, 18R, 20Z)‐variabilin hexadecanoate, (7E, 12E, 18R, 20Z)‐variabilin 10‐methylhexadecanoate, (7E, 12E, 18R, 20Z)‐variabilin 15‐methylhexadecanoate, (7E, 12E, 18R, 20Z)‐variabilin 14‐methylhexadecanoate, (7E, 12E, 18R, 20Z)‐variabilin 9‐octadecenoate, (7E, 12E, 18R, 20Z)‐variabilin octadecanoate, and (7E, 12E, 18R, 20Z)‐variabilin 2,11‐dimethyloctadecanoate, along with the recently described (7E, 12E, 18R, 20Z)‐variabilin 11‐methyloctadecanoate. The characterization of the new fatty acids (5Z, 9Z)‐22‐methyltricosadienoic and 2,11‐dimethyloctadecanoic acids is also described. The chemical structures were determined by extensive spectroscopic, chromatographic, and chemical analyses.
Low fat‐monounsaturated rich diets containing high‐oleic peanuts improve serum lipoprotein profiles
Authors: O’Byrne Dawn J. , Knauft David A. , Shireman Rachel B.
Published: November 2006

Postmenopausal hypercholesterolemic women are at risk for cardiovascular disease and are encouraged to follow low‐fat (LF) (≤30% energy) diets. However, these diets may have undesirable effects on high density lipoprotein cholesterol (HDL‐C), apolipoprotein A‐I (apo A‐I) and triglycerides, whereas diets high in monounsaturated fats do not. Twenty postmenopausal hypercholesterolemic women previously consuming high‐fat diets (34% energy) were placed on a low fat‐monounsaturated rich diet (LFMR: 26%, 14% energy, respectively) for 6 mon. Sixteen women already eating LF diets (24% energy) were also followed to monitor variations in serum lipids due to seasonal variations. Twenty‐five women successfully completed the study (LFMR=12, LF=13). Serum cholesterol decreased 10% (264 to 238 mg/dL, P≤0.01) and low density lipoprotein cholesterol (LDL‐C) decreased 12% (182 to 161 mg/dL, P≤0.01) in the LFMR group, but did not change in the LF group. The reduction in serum cholesterol in the LFMR group was greater than estimated by predictive formulas. Serum triglycerides and apo A‐I did not change in the LFMR group. A modest decrease in HDL‐C, HDL3‐C, and apolipoprotein B (apo B) occurred in both groups, but only the LFMR group showed a trend toward beneficial changes in LDL‐C/HDL‐C and apo A‐I/apo B ratios. Overall, the LFMR diet was well tolerated and resulted in an improved serum lipid and apolipoprotein profile.
Incorporation of n‐3 fatty acids into plasma lipid fractions, and erythrocyte membranes and platelets during dietary supplementation with fish, fish oil, and docosahexaenoic acid‐rich oil among healthy young men
Authors: Vidgren Helvi M. , Ågren Jyrki J. , Schwab Ursula , Rissanen Tiina , Hänninen Osmo , Uusitupa Matti I. J.
Published: November 2006

The effects of n‐3 fatty acid supplementation in the form of fresh fish, fish oil, and docosahexaenoic acid (DHA) oil on the fatty acid composition of plasma lipid fractions, and platelets and erythrocyte membranes of young healthy male students were examined. Altogether 59 subjects (aged 19–32 yr, body mass index 16.8–31.3 kg/m2) were randomized into the following diet groups: (i) control group; (ii) fish diet group eating fish meals five times per week [0.38±0.04 g eicosapentaenoic acid (EPA) and 0.67±0.09 g DHA per day]; (iii) DHA oil group taking algae‐derived DHA oil capsules (1.68 g/d DHA oil group taking algae‐derived DHA oil capsules (1.68 g/d DHA in triglyceride form); and (iv) fish oil group (1.33 g EPA and 0.95 g DHA/d as free fatty acids) for 14 wk. The fatty acid composition of plasma lipids, platelets, and erythrocyte membranes was analyzed by gas chromatography. The subjects kept 4‐d food records four times during the study to estimate the intake of nutrients. In the fish diet, in DHA oil, and in fish oil groups, the amounts of n‐3 fatty acids increased and those of n‐6 fatty acids decreased significantly in plasma lipid fractions and in platelets and erythrocyte membranes. A positive relationship was shown between the total n‐3 polyunsaturated fatty acids (PUFA) and EPA and DHA intake and the increase in total n‐3 PUFA and EPA and DHA in all lipid fractions analyzed. DHA was preferentially incorporated into phospholipid (PL) and triglyceride (TG) and there was very little uptake in cholesterol ester (CE), while EPA was preferentially incorporated into PL and CE. The proportion of EPA in plasma lipids and platelets and erythrocyte membranes increased also by DHA supplementation, and the proportion of linoleic acid increased in platelets and erythrocyte membranes in the DHA oil group as well. These results suggest retroconversion of DHA to EPA and that DHA also interferes with linoleic acid metabolism.
Conversion of 18∶3 Δ9cis, 12cis, 15trans in rat liver microsomes
Authors: Chardigny Jean‐Michel , Blond Jean‐Paul , Bretillon Lionel , Mager Estelle , Poullain Didier , Martine Lucy , Vatèle Jean‐Michel , Noël Jean‐Pierre , Sébédio Jean‐Louis
Published: November 2006

Several years ago, it was established that the Δ15 trans isomer of α‐linolenic acid is converted in vivo into fatty acids containing 20 and 22 carbons (geometrical isomers of eicosapentaenoic and docosahexaenoic acids). The present study focused on the in vitro Δ6 desaturation, the first step of the biosynthesis of the n‐3 long‐chain polyunsaturated fatty acids from 18:3n‐3. For that purpose, rat liver microsomes were prepared and incubated with radiolabeled 18∶3 Δ9cis, 12cis, 15cis (18∶3 c,c,c) or 18∶3 Δ9cis, 12cis, 15trans (18∶3c,c,t) under desaturation conditions. The data show that 18∶3c,c,t is converted at a lower rate compared with α‐linolenic acid. The product of conversion of 18∶3 c,c,t may be 18∶4 Δ6cis, 9cis, 12cis, 15trans resulting from a Δ6 desaturation of the trans substrate. Moreover, the conversion of radiolabeled 18∶3c,c,t was strongly decreased by the presence of 18∶3c,c,c (up to 48%) while the 18∶3c,c,t only slightly decreased the conversion of radiolabeled 18∶3c,c,c. Thus, the desaturation enzyme presented a higher affinity for the native all‐cis n‐3 substrate.
Increased hepatic β‐oxidation of docosahexaenoic acid, elongation of eicosapentaenoic acid, and acylation of lysophosphatidate in rats fed a docosahexaenoic acid‐enriched diet
Authors: Kanazawa Ayako , Shirota Yuri , Fujimoto Kenshiro
Published: November 2006

Rats were fed a diet supplemented with corn oil (n‐3 deficient), soy oil, or a mixture containing 8% 22∶6n‐3 ethyl ester for 6 wk. The hepatic capacities for the β‐oxidation and synthesis of 22∶6n‐3, in addition to the acylation of lysophosphatidate, were tested in vitro. In rats that were fed a 22∶6n‐3‐enriched diet, both the β‐oxidation of 22∶6n‐3 and elongation of 20∶5n‐3 were enhanced compared to those in rats fed the other diets. Acylation of lysophosphatidate was also enhanced in rats fed a 22∶6n‐3‐enriched diet, while the rate of dephosphorylation of phosphatidate was not changed. The amount of 22∶6n‐3 in the liver was much less than that consumed in a docosahexaenoic acid‐enriched diet. These results suggest that a significant amount of dietary 22∶6n‐3 was degraded via β‐oxidation, and that a portion of the retroconverted 20∶5n‐3 was recycled for the synthesis of 22∶6n‐3. The recycling of 20∶5n‐3 might contribute to the low level of 22∶6n‐3 in rats fed an n‐3‐deficient diet.
Oral acetylsalicylic acid induces biliary cholesterol secretion in the rat
Authors: Prigge William F. , Gebhard Roger L.
Published: November 2006

Several agents can alter biliary cholesterol secretion, critical for cholesterol excretion and gallstone formation. Although salicylate effects on bile formation and gallstones have been studied, biliary lipid secretion has not been measured during oral aspirin treatment. We examined whether oral acetylsalicylic acid affects bile lipid secretion. Three groups of young rats were fed chow for 3 wk. Two of the groups then received aspirin at either 1.67 or 3.33 g/kg diet for 4 d. Serum, hepatic, and bile lipids were measured, as were enzymes of cholesterol synthesis and esterification. With oral aspirin, bile cholesterol secretion increased by 42% and hepatic cholesteryl ester content decreased by 40%. Serum cholesterol and hepatic free cholesterol did not change. To evaluate mechanisms of the cholesterol hypersecretion, hypothyroid animals fed low‐fat or fish oil diets and repleted with triiodothyronine were also studied. Aspirin stimulated cholesterol secretion to a degree similar to triiodothyronine. An additive response was seen in fish oilfed rats. Aspirin did not appear to have a primary action on 3‐hydroxy‐3‐methylglutaryl‐CoA reductase or acyl CoA:cholesterol acyltransferase activities, and had no direct effect on esterification of cholesterol by isolated hepatocytes. Aspirin may directly increase cholesterol transport into bile or have cell membrane effects which alter cholesterol transport. It remains to be determined whether the observed alterations in bile cholesterol secretion are specific to the rat or also apply to humans.
An improved synthesis of taurine‐ and glycine‐conjugated bile acids
Authors: Momose Toshiaki , Tsubaki Takayuki , Iida Takashi , Nambara Toshio
Published: November 2006

A simple and efficient method for the synthesis of taurine‐ and glycine‐conjugated bile acids is described. The condensation reaction was achieved by the simple mixing of unconjugated bile acid (1.0 eq.), taurine (2.0 eq.) (or glycinate ester), diethyl phosphorocyanidate (1.2 eq.) in the presence of triethylamine at room temperature for 30–60 min. Sample clean‐up was effected by the use of a prepacked Sep‐Pak C18 cartridge for reversed‐phase solid extraction or by direct recrystallization, yielding the desired taurine and glycine conjugates in 89–93 and 92–96% isolated yields, respectively.
A synthesis of 4‐hydroxy‐2‐trans‐nonenal and 4‐(3H) 4‐hydroxy‐2‐trans‐nonenal
Authors: Chandra Animesh , Srivastava Satish K.
Published: November 2006

4‐Hydroxy‐2‐trans‐nonenal, the most abundant and toxic unsaturated aldehyde generated during membrane lipid peroxidation, was synthesized starting from fumaraldehyde dimethyl acetal. In the first step of the synthesis, the fumaraldehyde dimethyl acetal was partially hydrolyzed using amberlyst catalyst to obtain the monoacetal. The 4‐hydroxy‐2‐trans‐nonenal was synthesized by the Grignard reaction of the fumaraldehyde monoacetal with 1‐bromopentane. 4‐Hydroxy‐2‐trans‐nonenal, obtained as its dimethylacetal, was oxidized to its corresponding 4‐keto derivative using pyridinium chlorochromate buffered with sodium acetate as the oxidizing agent. 4‐(3H) 4‐Hydroxy‐2‐trans‐nonenal was obtained in one step by the sodium borotriteride reduction of the 4‐keto derivative.
Chromogenic assay for phospholipase D from Streptomyces chromofuscus: Application to the evaluation of substrate analogs
Authors: Hergenrother Paul J. , Haas Michelle K. , Martin Stephen F.
Published: November 2006

A rapid and convenient chromogenic assay for phospholipase D from Streptomyces chromofuscus (PLDSc) has been developed that converts the choline generated from the enzyme‐catalyzed hydrolysis of phospholipids into a chromogenic dye. By quenching the reaction with EDTA at defined times, an initial rate curve is produced from which a kcat and Km can be readily derived. This assay has been applied to the biological evaluation of several substrate analogs, all of which appear to be activators rather than substrates or inhibitors of this enzyme. Performing the assay in 96‐well microtiter plates allows for the easy screening of potential effectors of this enzyme.
Recombinant antibody fragments that detect enoyl acyl carrier protein reductase in Brassica napus
Authors: Ziegler Angelika , Macintosh Sybil M. , Torrance Lesley , Simon William , Slabas Antony R.
Published: November 2006

Purified Brassica napus enoyl acyl carrier protein reductase (ENR) was used to select specific antibodies from a library of antibody fragments, single‐chain Fv (scFv), displayed on filamentous phage. Analysis of the selected clones by BstNl fingerprinting and nucleotide sequencing showed that the scFv were derived from three different human VH germline genes. The binding specificities were confirmed by Western blots and ELISA. The scFv preparations reacted with B. napus ENR, but not with β‐keto reductase, nor enoyl reductase from Escherichia coli. Analysis of fragments generated by CNBr treatment indicates that the scFv 3.13 recognizes an epitope located within the n‐terminal 80 amino acids of the enzyme molecule. The scFv were used to detect ENR directly in extracts of B. napus seeds.
Use of cyclodextrin to deliver lipids and to modulate apolipoprotein B‐100 production in HepG2 cells
Authors: Peluso Michael R. , Dixon Joseph L.
Published: November 2006

2‐Hydroxypropyl‐β‐cyclodextrin (cyclodextrin), cyclodextrin‐solubilized oleate, and cyclodextrin‐solubilized cholesterol were used to modulate proteolysis and secretion of newly‐synthesized apolipoprotein B‐100 (apoB) in HepG2 cells. Following cyclodextrin and lipid treatments, cells were pulse‐labeled with [3H] leucine, and quantitative immunoprecipitation was used to measure apoB synthesis, apoB secreted into the medium, and the cellular content of undegraded apoB that was not secreted. Three‐hour treatment with cyclodextrin‐solubilized oleate (0.2 mM) increased secreted apoB from 4% (control cells) to 32% and cellular undegraded apoB from 15% (control cells) to 64% of apoB synthesized, which is consistent with earlier studies using bovine serum albumin to complex exogenous oleate. Prolonged daily (4 d or more) administration of 0.5% (3.5 mM) cyclodextrin with medium containing 10% fetal bovine serum increased the secretion of nascent apoB from 5–10% (control) to 17–28% and cellular undegraded apoB from 15–20% (control) to 25–31% of apoB synthesized, respectively. Subsequent administration of cyclodextrin‐solubilized cholesterol (10–40 μg) for only 3 h reversed the cyclodextrin‐mediated increase in apoB secretion. The application of 0.5% cyclodextrin to HepG2 cells can rapidly (within minutes) stimulate cholesterol efflux, and transiently (over a 1–2 d period) increase cholesterol synthesis. In the current studies, the cyclodextrin‐mediated increase in cholesterol synthesis was not concurrent with the increase in apoB secretion. However, prolonged (15 d) administration of cyclodextrin was shown to increase the cellular free cholesterol concentration by 25–41%, reduce the cellular triglyceride concentration by 59% and increase apoB secretion 3‐ to 4‐fold, without affecting the cellular cholesteryl ester concentration. In comparison, 14‐d treatment with cyclodextrin‐solubilized cholesterol (20 μg/mL) followed by 1‐d equilibration without cholesterol was shown to increase the cellular free cholesterol and cholesteryl ester concentrations by 76% and 10‐fold, respectively, although apoB secretion was not affected. It is hypothesized that chronic daily administration of 0.5% cyclodextrin increased the cellular cholesterol concentration and flux in discrete putative regulatory compartment which “shielded” nascent apoB from rapid proteolysis and facilitated apoB secretion. In conclusion, cyclodextrin was used independently and in combination with cholesterol or oleate to modulate apoB proteolysis and secretion. We speculate that subcellular changes in cholesterol concentration and flux may modulate apoB production in HepG2 cells.
Characterization of polar and nonpolar seed lipid classes from highly saturated fatty acid sunflower mutants
Authors: Álvarez‐Ortega Rosario , Cantisán Sara , Martínez‐Force Enrique , Garcés Rafael
Published: November 2006

The seed lipids from five sunflower mutants, two with high palmitic acid contents, one of them in high oleic background, and three with high stearic acid contents, have been characterized. All lipid classes of these mutant seeds have increased saturated fatty acid content although triacylglycerols had the highest levels. The increase in saturated fatty acids was mainly at the expense of oleic acid while linoleic acid levels remained unchanged. No difference between mutants and standard sunflower lines used as controls was found in minor fatty acids: linolenic, arachidic, and behenic. In the high‐palmitic mutants palmitoleic acid (16∶1n−7) and some palmitolinoleic acid (16∶2n−7, 16∶2n−4) also appeared. Phosphatidylinositol, the lipid with the highest palmitic acid content in controls, also had the highest content of palmitic or stearic acids, depending on the mutant type, suggesting that saturated fatty acids are needed for its physiological function. Positional analysis showed that mutant oils have very low content of saturated fatty acids in the sn‐2 position of triacylglycerols, between the content of olive oil and cocoa butter.
Lipids and ultrastructure of Thraustochytrium sp. ATCC 26185
Authors: Weete J. D. , Kim H. , Gandhi S. R. , Wang Y. , Dute R.
Published: November 2006

As a representative of a genus with species considered to be potential commercial producers of the nutritionally important polyunsaturated fatty acid docosahexaenoic acid (DHA), Thraustochytrium sp. ATCC 26185 was investigated to determine its potential for DHA production and lipid composition. Cells from liquid shake cultures contained 32% (w/w) lipid, 18% of which was nonsaponifiable lipid. The major saturated fatty acids (14∶0 and 16∶0) comprised up to 59% of the total fatty acids, and DHA was up to 25% after 6 d incubation. Squalene represented 63% of the nonsaponifiable lipid, and cholesterol composed 41% of the total sterols. The phospholipids expected for eucaryotic microbes were detected with phosphatidylcholine as the major phospholipid at 76% of the total. The ultrastructure of this species was similar to other Thraustochytrium species except that the cells did not have surface scales and they contained unusual membrane‐like structures that appeared to be associated with oil formation.
On the interrelationship between hepatic carnitine, fatty acid oxidation, and triglyceride biosynthesis in nephrosis
Authors: AL‐Shurbaji Ayman , Berglund Lars , Berge Rolf K. , Cederblad Gitten , Humble Elisabet
Published: November 2006

The nephrotic syndrome is associated with disturbances in plasma lipid pattern and metabolism. However, the reason for these perturbations is poorly understood. In the present study, we have investigated hepatic triglyceride metabolism in puromycin aminonucleoside‐induced nephrotic syndrome in rats. Nephrotic rats displayed a 70% increase in hepatic triglyceride levels compared to controls (16.9±1.6 vs. 9.8±0.6 μmol/g liver; means±SEM, P<0.01). The capacity for hepatic mitochondrial β‐oxidation of fatty acids was substantially elevated (80%). This was associated with a rise in the liver content of the fatty acid carrier carnitine (1.24±0.06 vs. 0.85±0.07 μmol/g dry weight, P<0.05). A positive correlation between the levels of acetylcarnitine and acetyl‐CoA was found in normal as well as in nephrotic rats, implying that carnitine plays an important role as an acetyl group acceptor in the liver under normo‐ and hyperlipidemic conditions. Changes in carnitine levels seem to be tightly coupled to the rate of fatty acid oxidation. There was a significant elevation in the activity of phosphatidate phosphohydrolase (E.C. 3.1.3.4) in liver microsomes from nephrotic rats (1.07±0.09 vs. 0.81±0.04 nmol/min·mg protein, P<0.02). Hepatic very low density lipoprotein (VLDL)‐triglyceride secretion rate was 18% higher in nephrotic rats than in controls. The results demonstrate a deranged hepatic triglyceride metabolism in nephrosis, with an increased hepatic triglyceride biosynthesis, a sizable accumulation of hepatic triglycerides, and only a modest increase in VLDL triglyceride secretion. In addition, mitochondrial β‐oxidation of fatty acids was enhanced, associated with an increased availability of carnitine.
Effect of conjugated linoleic acid on body composition in mice
Authors: Park Yeonhwa , Albright Karen J. , Liu Wei , Storkson Jayne M. , Cook Mark E. , Pariza Michael W.
Published: November 2006

The effects of conjugated linoleic acid (CLA) on body composition were investigated. ICR mice were fed a control diet containing 5.5% corn oil or a CLA‐supplemented diet (5.0% corn oil plus 0.5% CLA). Mice fed CLA‐supplemented diet exhibited 57% and 60% lower body fat and 5% and 14% increased lean body mass relative to controls (P<0.05). Total carnitine palmitoyltransferase activity was increased by dietary CLA supplementation in both fat pad and skeletal muscle; the differences were significant for fat pad of fed mice and skeletal muscle of fasted mice. In cultured 3T3‐L1 adipocytes CLA treatment (1×10−4 M) significantly reduced heparin‐releasable lipoprotein lipase activity (−66%) and the intracellular concentrations of triacylglyceride (−8%) and glycerol (−15%), but significantly increased free glycerol in the culture medium (+22%) compared to control (P<0.05). The effects of CLA on body composition appear to be due in part to reduced fat deposition and increased lipolysis in adipocytes, possibly coupled with enhanced fatty acid oxidation in both muscle cells and adipocytes.
Short‐term effects of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase inhibitor on cholesterol and bile acid synthesis in humans
Authors: Yoshida Tadashi , Honda Akira , Shoda Junichi , Abei Masato , Matsuzaki Yasushi , Tanaka Naomi , Miyazaki Hiroshi , Osuga Toshiaki
Published: November 2006

Competitive inhibitors of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase improve hypercholesterolemia. However, reports about the effects of these agents on bile acid synthesis, the metabolic pathway of cholesterol, are conflicting. We studied the short‐term effect of one of these agents, pravastatin, on bile acid synthesis. Six male volunteers were given 40 mg of pravastatin. Plasma mevalonate level (which reflects cholesterol synthesis) and 7α‐hydroxy‐4‐cholesten‐3‐one level 9which reflects bile acid synthesis) were measured every 2 h for 8 h. These plasma levels were compared to those of the same volunteers without pravastatin. Plasma mevalonate level after 2 h was lower than control (3.0 ± 1.1 ng/ml vs. 6.7 ± 2.5, mean ±SD; P<0.05). This decrease continued for 8 h (2.5 ± 0.8 vs. 5.2 ± 1.5; P<0.05). On the other hand, plasma 7α‐hydroxy‐4‐cholesten‐3‐one level did not change until after 6 h; then at 8 h it was lower than control (15.7 ± 11.8 ng/mL vs. 24.7 ± 16.9; P <0.05). According to three‐way layout analysis of variance, mevalonate level was influenced by both pravastatin treatment (P<0.01) and time‐course (P<0.01). On the other hand, the 7α‐hydroxy‐4‐cholesten‐3‐one level was affected by both individual difference (P<0.01) and time course (P<0.01), but pravastatin treatment did not influence this compound. This indicates that bile acid synthesis was not influenced by pravastatin, although cholesterol synthesis was inhibited. The shortterm inhibition of cholesterol synthesis did not affect bile acid synthesis.
Oxidation of cholesterol in synaptosomes and mitochondria isolated from rat brains
Authors: Vatassery Govind T. , Quach Hung T. , Smith W. Ed , Ungar Frank
Published: November 2006

Cholesterol and α‐tocopherol oxidations were studied in brain subcellular fractions isolated from cerebral hemispheres of 4‐month‐old, male Fischer 344 rats. The fractions were suspended in buffered media (pH 7.4, 37°C) and oxidized by adding (i) ferrous iron (Fe2+) with or without ascorbate or (ii) peroxynitrite (an endogenous oxidant produced by the reaction of superoxide and nitric oxide). Treatment of subcellular fractions with Fe2+ in the presence or absence of ascorbate produced primarily 7‐keto‐ and 7‐hydroxy‐cholesterols and small amounts of 5α,6α‐epoxycholesterol. Since brain contains high levels of ascorbate, any release of iron could result in oxysterol formation. Peroxynitrite oxidized α‐tocopherol but not cholesterol. Hence, the toxicity of peroxynitrite or nitric oxide could not be due to cytotoxic oxysterols. When synaptosomes were incubated for 5 min in the presence of 0.5 to 2 μM Fe2+ and ascorbate, α‐tocopherol was oxidized while cholesterol remained unchanged. Thus, α‐tocopherol is functioning as an antioxidant, protecting cholesterol. Diethylenetriaminepentaacetic acid blocked production of oxysterols, whereas citrate, ADP and EDTA dit not. A significant percentage of mitochondrial cholesterol was oxidized by treatment with Fe2+ and ascorbate. Hence, mitochondrial membrane properties dependent on cholesterol could be particularly susceptible to oxidation. The oxysterols formed were retained within the membranes of synaptosomes and mitochondria. The 7‐oxysterols produced are known to be inhibitors of membrane enzymes and also can modify membrane permeability. Hence, oxysterols may play an important role in brain tissue damage during oxidative stress.