bluuebunny/crossref_metadata_2025 huggingface dataset:
Tissue‐selective inhibition of sterol synthesis in mice by pravastatin sodium after a single or repeated oral administrations
Authors: Koga Teiichiro , Kikuchi Takashi , Miyazaki Atsuhiro , Koike Hiroyuki
Published: November 2006

Pravastatin, an inhibitor of 3‐hydroxy‐3‐methylglutaryl CoA (HMG‐CoA) reductase, exhibits liver‐selectivity in inhibiting sterol synthesis, when administered as a single oral dose to mice or rats, whereas lovastatin and simvastatin do not. This may be due to the fact that pravastatin is distributed intracellularly, to a large extent, in the liver and extracellularly in nonhepatic tissues. In the present study, we examined whether the difference in liver‐selectivity among these three HMG‐CoA reductase inhibitors observed in single‐dose studies was preserved after repeated oral administrations of drugs to mice.De novo sterol synthesis in different tissues of mice was examinedin vivo three hours after the last dose of drug by measuring incorporation of intraperitoneally injected [14C]acetate into total sterols. Pravastatin administered orally for 11 consecutive days at 5 and 10 mg/kg exhibited a greater liver‐selectivity than lovastatin and simvastatin: sterol synthesis was inhibited more than 60% in the liver by all three drugs, whereas that in nonhepatic tissues was inhibited less than 10% by pravastatin and more than 30% by lovastatin and simvastatinin in most of the nonhepatic tissues examined. Pravastatin administered orallyfor 11 consecutive days at 10 mg/kg caused more selective inhibition of sterol synthesis in liverex vivo than two other inhibitors at the same dose. Pravastatin inhibitedde novo sterol synthesis from [14C]acetate into sterol fraction in the liver slicesin vitro, but minimally in those of the spleen and testis, whereas lovastatin and simvastatin inhibited in those of all three tissues. Since the drug concentrations determined in the same tissue samples of the liver, spleen, and testis were almost comparable among the three drugs, it was suggested that the cellular distribution profiles of pravastatin observed in a single‐dose study were preserved in the multiple‐dose study. We conclude that the difference in tissue‐selectivity between pravastatin and the other two inhibitors to inhibit sterol synthesis in mice is maintained, regardless of the duration of administration.
Some new aspects of isoprenoid biosynthesis in plants—A review
Authors: Bach Thomas J.
Published: November 2006

Plants are capable of synthesizing a myriad of isoprenoids and prenyl lipids. Much attention has been focused on 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMGR), the enzyme that synthesizes mevalonate and is generally considered responsible for the regulation of substrate flux to isoprenoids. In contrast to vertebrates, where there seems to exist only one HMGR gene, in plants a small family of isogenes appears differentially expressed in regard to location and time. Much less is known in plants about the preceding steps,viz. the conversion of acetyl‐CoA to HMG‐CoA. An enzyme system has been isolated from radish that can catalyze this transformation, and which shows some unusual propertiesin vitro. The intracellular localization of the early steps of isoprenoid biosynthesis in plant cells is still a matter of debate. The various observations and hypotheses derived from incorporation and inhibition studies are somewhat contradictory, and an attempt is being made to rationalize various findings that do not at first seem compatible. There are good arguments in favor of an exclusively cytoplasmic formation of isopentenyl pyrophosphate (IPP) via mevalonic acid, but other studies and observations suggest an independent formation in plastids. Other possibilities are being considered, such as the existence of independent (compartmentalized) biosynthetic pathways of IPP formationvia the socalled Rohmer pathway. Substrate channeling through the formation of end product‐specific multienzyme complexes (metabolons) with no release of substrate intermediates will also be discussed.
Developmental regulation of sterol biosynthesis inZea mays
Authors: Guo De‐an , Venkatramesh M. , Nes W. David
Published: November 2006

Sixty‐one sterols and pentacyclic triterpenes have been isolated and characterized by chromatographic and spectral methods fromZea mays (corn). Several plant parts were examined; seed, pollen, cultured hypocotyl cells, roots, coleoptiles (sheaths), and blades. By studying reaction pathways and mechanisms on plants fed radiotracers ([2‐14C]mevalonic acid, [2‐14C]acetate, and [2‐3H]acetate), and stable isotopes (D2O), we discovered that hydroxymethylglutaryl CoA reductase is not “the” rate‐limiting enzyme of sitosterol production. Additionally, we observed an ontogenetic shift and kinetic isotope effect in sterol biosynthesis that was associated with the C‐24 alkylation of the sterol side chain. Blades synthesized mainly 24α‐ethyl‐sterols, sheaths synthesized mainly 24‐methyl‐sterols, pollen possessed an interrupted sterol pathway, accumulating 24(28)‐methylene‐sterols, and germinating seeds were found to lack an activede novo pathway. Shoots, normally synthesizing (Z)‐24(28)‐ethylidine‐cholesterol, after incubation with deuterated water, synthesized the rearranged double‐bond isomer, stigmasta‐5,23‐dien‐3β‐ol. Examination of the mass spectrum and1H nuclear magnetic resonance spectrum of the deuterated 24‐ethyl‐sterol indicated the Bloch‐Cornforth route originating with acetyl‐CoA and passing through mevalonic acid to sterol was not operative at this stage of development. An alternate pathway giving rise to sterols is proposed.
Cloning of the late genes in the ergosterol biosynthetic pathway ofSaccharomyces cerevisiae—A review
Authors: Lees N. D. , Skaggs B. , Kirsch D. R. , Bard M.
Published: November 2006

Research on the ergosterol biosynthetic pathway in fungi has focused on the identification of the specific sterol structure required for normal membrane structure and function and for completion of the cell cycle. The pathway and its end product are also the targets for a number of antifungal drugs. Identification of essential steps in ergo‐sterol biosynthesis could provide new targets for the development of novel therapeutic agents. Nine of the eleven genes in the portion of the pathway committed exclusively to ergosterol biosynthesis have been cloned, and their essentiality for aerobic growth has been determined. The first three genes;ERG9 (squalene synthase),ERG1 (squalene epoxidase), andERG7 (lanosterol synthase), have been cloned and found to be essential for aerobic viability since their absence would result in the cell being unable to synthesize a sterol molecule. The remaining eight genes encode enzymes which metabolize the first sterol, lanosterol, to ultimately form ergosterol. The two earliest genes,ERG11 (lanosterol demethylase) andERG24 (C‐14 reductase), have been cloned and found to be essential for aerobic growth but are suppressed by mutations in the C‐5 desaturase (ERG3) gene andfen1 andfen2 mutations, respectively. The remaining cloned genes,ERG6 (C‐24 methylase),ERG2 (D8Æ7 isomerase),ERG3 (C‐5 desaturase), andERG4 (C‐24(28) reductase), have been found to be nonessential. The remaining genes not yet cloned are the C‐4 demethylase and the C‐22 desaturase (ERG5).
Biochemical and physiological effects of sterol alterations in yeast—A review
Authors: Parks Leo W. , Smith Steven J. , Crowley James H.
Published: November 2006

Considerable progress has been made in the selection and characterization of mutants that are defective in the synthesis of ergosterol in the yeast,Saccharomyces cerevisiae. Mutations in nearly every step of the yeast sterol biosynthetic pathway have been induced and selected. These mutants have been used to elucidate the sequential order of steps in sterol synthesis, to study the mode of action of antifungal agents and to determine the method of resistance of some pathogenic fungi, and to answer questions on the role of sterols in general cell biology. Physiological examination of ergosterol null mutants, lacking all biochemical activity attributed to the particular gene, supports a role for ergosterol in a number of critical functions in the organism. Among the physiological functions attributed to ergosterol are sparking and bulking requirements, involvement in amino acid and pyrimidine transport, resistance to antifungal agents and certain cations, and a requirement for respiratory activity. Those genetic null alleles discussed in this review areerg24, lacking the ability to reduce the Δ14 double bond;erg6, unable to methylate C‐24; anderg3, defective in the C‐5 desaturase. The different biochemical activities that are disrupted in the ergosterol mutants support a role for ergosterol in a number of critical functions in yeast.
Identification of the active site of vertebrate oxidosqualene cyclase
Authors: Abe Ikuro , Prestwich Glenn D.
Published: November 2006

Active site mapping of rat liver oxidosqualene cyclase (OSC), a 78 kDa membrane‐bound enzyme, was carried out using the mechanism‐based irreversible inhibitor, [3H]29‐methylidene‐2,3‐oxidosqualene. The amino acid sequence of the radiolabeled CNBr peptide fragment showed unexpectedly high similarity to the yeast OSC, plant OSC, and bacterial squalene cyclases. Further, radio analysis established that the two adjacent Asp residues in the highly conserved region (Asp‐Asp‐Thr‐Ala‐Glu‐Ala, or DDTAEA) were equally labeled by the irreversible inhibitor. This result provided the first information on the structural details of the active site of OSC, and showed for the first time the ancient lineage of this vertebrate enzyme to ancestral eukaryotic and prokaryotic cyclases. Interestingly, the covalently‐modified DDXX(D/E) sequence of rat liver OSC showed surprising similarity to the putative allylic diphosphate binding site sequence of other terpene cyclases and prenyl transferases. The Asp‐rich motif may act as a point charge to stabilize incipient cationic charge.
Biosynthesis and distribution of insect‐molting hormones in plants—A review
Authors: Adler John H. , Grebenok Robert J.
Published: November 2006

Insect‐molting hormones, phytoecdysteroids, have been reported to occur in over 100 plant families. Plants, unlike insects, are capable of the biosynthesis of ecdysteroids from mevalonic acid, and in several cases the biosynthesis of phytoecdysteroids was also demonstrated to proceedvia sterols.Spinacia oleracea (spinach) biosynthesizes polypodine B and 20‐hydroxyecdysone, which is the predominant insect‐molting hormone found in plant species. The onset of ecdysteroid production in spinach requires the appropriate ontogenetic development within the plant, which is related to leaf development. In spinach, lathosterol is the biosynthetic precursor to ecdysone and 20‐hydroxyecdysone. Phosphorylated ecdysteroid intermediates, particularly ecdysone‐3‐phosphate, are required during biosynthesis. Polyphosphorylated forms of ecdysteroids are putative regulatory components of the pathway. During spinach development, the 20‐hydroxyecdysone is transported from the sites of biosynthesis to the apical regions. An analysis of the physiological data available suggests that different species may synthesize ecdysteroids in various organs and distribute these ecdysteroids to other sites. Annual plants appear to concentrate ecdysteroids in the apical regions, including flowers and seeds. Perennial plants may recycle their ecdysteroids between their deciduous and their perennial organs over the growing season. Further investigations of ecdysteroid biosynthesis and physiology within plants will be required before an acceptable system can be designed to test phytoecdysteroid effectivenessin vivo against insect herbivory.
Sterol metabolism in the tobacco hornworm,Manduca sexta—A review
Authors: Svoboda James A. , Weirich Gunter F.
Published: November 2006

A number of intermediates involved in the dealkylation and conversion of the major C28 and C29 phytosterols to cholesterol in insects were first isolated and identified in studies with the tobacco hornworm,Manduca sexta, carried out in our laboratory. We also investigated the effects of a variety of known sterol metabolism inhibitors inManduca, particularly those affecting the Δ24‐sterol reductase enzyme, and synthesized and tested a number of new inhibitors as well. In‐depth studies of ecdysteroids inManduca during embryogenesis and during pupal‐adult development provided new information on molting hormone content, biosynthesis, and metabolism. In addition, this insect has been utilized in the study of three specific enzyme systems of ecdysteroid metabolism, namely 20‐monooxygenase, 3‐epimerase, and phosphotransferase, which are critical to activation and deactivation of molting hormones in insects.
Fatty chain compositon of ether and ester glycerophospholipids in the Japanese oysterCrassostrea gigas (Thunberg)
Authors: Koizumi Chiaki , Jeong Bo Young , Ohshima Toshiaki
Published: November 2006

The fatty chain compositions of 1‐O‐alk‐1′‐enyl‐2‐acyl, 1‐0‐alkyl‐2‐acyl, and 1,2‐diacyl glycerophospholipids of the Japanese oysterCrassostrea gigas (Thunberg) were investigated. Major fatty chains in thesn‐1 position of 1‐alk‐1′‐enyl‐2‐acyl ethanolamine phospholipids (EPL) were 18∶0 (64.7%) and 20∶1 (11.1%). Majorsn‐1 chains of alkenylacyl choline phospholipids (CPL) were 18∶0 (63.3%) and 16∶0 (22.2%). In the case of 1‐alkyl‐2‐acyl EPL, the predominant fatty chains in thesn‐1 position were 18∶0 (51.5%), 16∶0 (16.0%) and 20∶1 (12.5%); in the case of 1‐alkyl‐2‐acyl CPL, the majorsn‐1 chains were 16∶0 (44.0%) and 14∶0 (23.4%). Saturated fatty chains were predominant in both EPL and CPL. Prominent fatty acids in thesn‐2 position of the alkenylacyl EPL were 22∶6n−3 (29.0%), 20∶5n−3 (19.0%) and 22∶2 NMID (non‐methylene interrupted dienes, 16.6%) contributing to about 65% of the total fatty acids, while alkenylacyl CPL was rich in the saturated acids 16∶0 (32.0%) and 18∶0 (9.2%). In the alkylacyl EPL, 16∶0, 18∶1n−9, 18∶0 and 16∶1n−7 were prominentsn‐2 fatty acids and accounted for 30.6%, 10.0%, 9.8%, and 8.3%, respectively. Polyunsaturated fatty acids were detected, but were present at extremely low percentages. Majorsn‐2 fatty acids in alkylacyl CPL were 16∶0 (25.4%), 22∶6n−3 (16.0%) and 20∶5n−3 (8.4%). The major fatty acids of diacyl EPL were 20∶5n−3 (22.3%), 16∶0 (17.9%), and 18∶0 (16.1%), and those of diacyl CPL were 16∶0 (30.4%), 20∶5n−3 (17.6%) and 18∶1n−7 (7.4%).
Enantiomer separations of mixtures of monoacylglycerol derivatives by HPLC on a chiral column
Authors: Takagi Toru , Ando Yasuhiro
Published: November 2006

High‐performance liquid chromatographic separations of monoacylglycerol enantiomeric mixtures as their di‐3,5‐dinitrophenylurethane derivatives were carried out on a chiral stationary phase, N‐(R)‐1‐(α‐naphthyl)ethylaminocarbonyl‐(S)‐valine bonded to silica gel. The conditions for the quantitative analysis ofsn‐1 andsn‐3 enantiomeric mixtures of six saturated 1‐monoacylglycerols having consecutive even carbon numbers and ofsn‐1 andsn‐3 enantiomeric mixtures of 1‐stearoyl‐, 1‐oleoyl‐, 1‐linoleoyl‐, and 1‐linolenoyl‐glycerols were obtained. The linear relationships of logarithmic retention volumes against the number of carbons and olefinic bonds for the enantiomer homologous series are discussed, and a new concept of enantiomer separation is presented.
Quantitation of vitamin K in human milk
Authors: Canfield Louise M. , Hopkinson Judy M. , Lima Anne F. , Martin Gail S. , Sugimoto Kyoto , Burr Jeanne , Clark Larry , McGee Daniel L.
Published: November 2006

A quantitative method was developed for the assay of vitamin K in human colostrum and milk. The procedure combines preparative and analytical chromatography on silica gel in a nitrogen atmosphere followed by reversed phase high performance liquid chromatography (HPLC). Two HPLC steps were used: gradient separation with ultraviolet (UV) detection followed by isocratic separation detected electrochemically. Due to co‐migrating impurities, UV detection alone is insufficient for identification of vitamin K. Exogenous vitamin K was shown to equilibrate with endogenous vitamin K in the samples. A statistical method was incorporated to control for experimental variability. Vitamin K1 was analyzed in 16 pooled milk samples from 7 donors and in individual samples from 15 donors at 1 month post‐partrum. Vitamin K1 was present at 2.94±1.94 and 3.15±2.87 ng/mL in pools and in individuals, respectively. Menaquinones, the bacterial form of the vitamin, were not detected. The significance of experimental variation to studies of vitamin K in individuals is discussed.
A simple device for the optimized isolation of fat cells
Authors: Meignen J. M. , Desnuelle C. , Boyer J.
Published: November 2006

We describe a simple cell isolation device that allows the separation and sequential collection of fat cells as they are released from adipose tissue fragments exposed to a collagenase preparation. This is done without interrupting the digestion process. Use of the device allows for appreciable lowering of the degree of cell damage and the extent to which larger cells are ruptured during the isolation process. Fat cells isolated by this technique elicit a higher level of metabolic activity as reflected by their improved response to the lipolytic action of epinephrine.
Lipids in gap junction assembly and function
Authors: Malewicz Barbara , Kumar V. V. , Johnson Ross G. , Baumann Wolfgang J.
Published: November 2006

Gap junctions (GJ) are important regulators of cellular function. They provide channels for the direct movement of small molecules between cells and thus control cell‐to‐cell transfer of metabolites and the transmission of various stimuli. Gap junctions have been shown to be involved in a multitude of cellular processes ranging from cell synchronization and neuronal function to cell differentiation and carcinogenesis. Much knowledge has been gained in recent years concerning the structure and molecular organization of GJ proteins; yet, the mechanisms that control and modulate gap junction assembly and function are still not well understood. Although it is quite apparent that the GJ proteins assemble in the lipid milieu of the plasma membrane, and that the cluster of proteins assembled in the junction do function in a lipid environment, there is a general paucity of information on the role of lipids in the gap junction assembly process and in the function of gap junctions.

The present review is a comprehensive account of current knowledge on gap junction lipids. We also discuss what is known to date on the involvement of lipids in gap junction formation. Special emphasis is being placed on the potential role of membrane cholesterol in gap junction assembly and function.
Effect of temperature on the incorporation into phospholipid classes and metabolismvia desaturation and elongation of n−3 and n−6 polyunsaturated fatty acids in fish cells in culture
Authors: Tocher Douglas R. , Sargent John R.
Published: November 2006

The incorporation of 18∶2n−6, 18∶3n−3, 20∶4n−6 and 20∶5n−3 was greater at 10°C than at 22°C in Atlantic salmon (AS), rainbow trout (RTG‐2) and turbot (TF) cells. However, there were generally no significant differences between the amount of incorporation of all four polyunsaturated fatty acids (PUFA) into total lipid within a cell type at either 22°C or 10°C. The distributions of the PUFA between individual phospholipid classes at 22°C was essentially the same in AS and TF cells—with the C18 PUFA the order of incorporation in these cells was phosphatidylcholine (PC) > phosphatidylethanolamine (PE) > phosphatidic acid/cardiolipin (PA/CL); with 20∶4n−6 the order was PE and phosphatidylinositol (PI)>PC; with 20∶5n−3, PE>PC. In RTG‐2 cells at 22°C the distributions of the C18 PUFA were similar to the other cell lines, but with 20∶4n−6 the order was PC>PI>PE, and with 20∶5n−3 it was PC>PE. At 10°C the incorporation of C18 PUFA into PC increased and into PE and PA/CL decreased, in general, in all cell lines. Incorporation of 20∶5n−3 into PC and PE was increased and decreased at 10°C, respectively, in AS and TF cells, whereas in RTG‐2 cells the changes at 10°C were opposite i.e., increased in PE and decreased in PC. With 20∶4n−6, incorporation into PC at 10°C was increased in all cell lines with decreased incorporation into PI in AS and RTG‐2 cells and into PE in AS and TF cells, whereas incorporation of 20∶4n−6 into PE increased in RTG‐2 cells. The metabolismvia desaturation and elongation of the n−3 PUFA was greater than that of the equivalent n−6 PUFA in all cell lines, irrespective of temperature. There was less conversion of the C18 PUFA at 10°C than at 22°C in RTG‐2 and TF cells, but the conversion of 18∶3n−3 by AS cells was increased at 10°C. Temperature had no effect on the conversion of the C20 PUFA.
Phospholipid and fatty acid composition of frog (Rana esculenta) liver—a circannual study
Authors: Scapin S. , Baldini P. , Luly P.
Published: November 2006

Liver lipid composition of the frogRana esculenta was examined on a circannual basis. In particular, phospholipid and cholesterol content, relative phospholipid distribution, and fatty acid patterns have been studied. Seasonal acclimatization is associated with significant modifications of phospholipid content and of the relative proportion of phospholipid classes, while cholesterol level is unchanged throughout the year. In regard to the fatty acid composition of total phospholipids as well as of the four major phospholipid classes—phosphatidylcholine (PC); phosphatidylethanolamine (PE); sphingomyelin (SM); phosphatidylserine (PS)—it appears that the liver of “summer animals” is characterized by a higher unsaturation index due to a decrease of saturated fatty acids and to an increased content of n−3 and n−6 polyunsaturated fatty acids. The results suggest that relevant compositional changes occur mainly in spring and autumn: these changes could be interpreted as being the result of both a nutritionally‐ and thermally‐induced seasonal adaptation directed toward the preservation of membrane‐associated physiological activities that are linked to the transition from the active to the inactive state of the animal.
The association of lysophosphatidylcholine with isolated cardiac myocytes
Authors: Man Ricky Y. K. , Kinnaird Anne A. A. , Bihler Ivan , Choy Patrick C.
Published: November 2006

The ability of exogenous lysophosphatidylcholine to produce electrophysiological derangements and cardiac arrhythmias in the heart has been documented. The action of lysophosphatidylcholine is thought to be mediated via its association with the membrane. The present study examined the nature of the association of lysophosphatidylcholine with isolated rat myocyte membrane. The association was studied by incubating myocytes in a lysophosphatidylcholine‐containing medium. The association of lysophosphatidylcholine with the myocyte sarcolemma was not affected by palmitic acid and glycerophosphocholine but was reduced by platelet‐activating factor (PAF). The addition of albumin (5 mg/mL) at the end of the incubation period effectively removed the lysophosphatidylcholine from the myocytes. Our results suggest that most of the lysophosphatidylcholine in isolated myocytes was associated preferentially with the outer leaflet of the myocyte sarcolemma. This type of association might be responsible for the lysophosphatidylcholine‐induced electrophysiological alterations in the heart.
Influence of dietary linoleic acid andtrans fatty acids on the fatty acid profile of cardiolipins in rats
Authors: Høy Carl‐Erik , Hølmer Gunhild
Published: November 2006

Cardiolipins (CL) have unique fatty acid profiles with generally high levels of polyunsaturated fatty acids, primarily 18∶2n−6, and low levels of saturated fatty acids. In order to study the effect of dietary fatty acid isomers on the fatty acid composition of cardiolipins, rats were fed partially hydrogenated marine oils (HMO), rich in 16∶1, 18∶1, 20∶1, and 22∶1 isomeric fatty acids, supplemented with linoleic acid at levels ranging from 1.9% to 14.5% of total fat. Although the dietary fats contained 33%trans fatty acids, the levels oftrans fatty acids in CL were below 2.5% in all organs. The fatty acid profiles of cardiolipins of liver, heart, kidney and testes showed different responses to dietary linoleic acid level. In liver, the contents of 18∶2 reflected the dietary levels. In heart and kidney, the levels of 18∶2 also parallelled increasing dietary levels, but in all groups fed HMO, levels of 18∶2 were considerably higher than in the reference group fed palm oil. In testes, the 18∶2 levels were unaffected by the dietary level of 18∶2 and HMO.
Phospholipid fatty acid composition of various mouse tissues after feeding α‐linolenate (18∶3n−3) or eicosatrienoate (20∶3n−3)
Authors: Berger Alvin , German J. Bruce
Published: November 2006

The selective incorporation of dietary α‐linolenate (18∶3n−3) and its elongation product, eicosatrienoate (20∶3n−3), into various phospholipids (PL) of mouse liver, spleen, kidney, and heart, was examined in a two‐week feeding trial by assessing mol % changes in associated fatty acids. Mice were fed fat‐free AIN 76A diets modified with either 2 wt% safflower oil (control); 1% safflower and 1% linolenate; or 1% safflower and 1% eicosatrienoate. After linolenate or eicosatrienoate feeding, 20∶4n−6 was reduced by 36–50% in liver phosphatidylcholine (PC) and in liver and spleen phosphatidylethanolamine (PE). Linolenate was minimally incorporated into PL, but was desaturated and elongated to 20∶5n−3, 22∶5n−3, and 22∶6n−3, with notable differences in the quantity of these n−3 derivatives associated with different tissues and PL. Eicosatrientoate was uniquely incorporated into the cardiolipin (CL) pool of all organs. There was also considerable retroconversion of 20∶3n−3 to 18∶3n−3 (PC, PE). Dietary eicosatrienoate may therefore affect metabolism in diverse ways—20∶3n−3, which is retroconverted to 18∶3n−3, may provide substrate for 20∶5n−3 and 22∶6n−3 syntheses, whereas intact 20∶3n−3 may be incorporated into the CL pool. Acyl modifications of CL are known to affect the activity of key innermitochondrial enzymes, such as cytochrome c oxidase.
Synthesis and characterization of cytochrome P‐450 epoxygenase metabolites of eicosapentaenoic acid
Authors: VanRollins Mike
Published: November 2006

Eicosapentaenoic acid, a major component of fish oil extracts, was recently shown to be metabolized to vicinal diol regioisomers by renal and hepatic cytochrome P‐450 epoxygenases. The diol products were also found in the urine of people ingesting fish oil. In the present report, we developed a chemical method of making milligram amounts of the epoxide intermediates and their diol products. Eicosapentaenoic acid was reacted with 0.1 eq.m‐chloroperoxybenzoic acid for 15 min. After normal‐ and reverse‐phase high performance liquid chromatography plus capillary gas chromatography and electron impact mass spectrometry, the products were identified as 17,18‐cis‐epoxyeicosa‐5,8,11,14‐tetraenoic, 14,15‐cis‐epoxy‐eicosa‐5,8,11,17‐tetraenoic, 11,12‐cis‐epoxy‐eicosa‐5,8,14,17‐tetraenoic, 8,9‐cis‐epoxy‐eicosa‐5,11,14,17‐tetraenoic and 5,6‐cis‐epoxy‐eicosa‐8,11,14,17‐tetraenoic acids. The total epoxide yield from eicosapentaenoate was 10% per incubation. By reincubating (cycling) the unused substrate 10–20 times, the total epoxide yield could be increased to 66–88%. Epoxide regioisomers were not produced in equal amounts; epoxygenation was facilitated as the distance between the target double bond and carbomethoxy group increased. Upon hydrolyzing the epoxides, the gas‐chromatographic retention times and mass spectra of the diol products were found to match those of biological metabolites. In addition, each of these standards was rapidly and quantitatively synthesized in an amount (milligram) adequate for biological tests.
Nuclear magnetic resonance spectroscopic analysis of homoallylic andbis homoallylic substituted methyl fatty ester derivatives
Authors: Lie Ken Jie Marcel S. F. , Cheng K. L.
Published: November 2006

Using a combination of selective irradiation1H nuclear magnetic resonance experiments and two‐dimensional1H−13C correlation spectroscopy spectral analysis of homoallylic andbis homoallylic substituted (azido, acetoxy, chloro and oxo) fatty ester derivatives, the carbon shifts of the ethylenic carbon atoms were determined. In the case of methyl 12‐azido‐9Z‐octadecenoate (homoallylic), the carbon chemical shifts of the ethylenic C‐9 and C‐10 carbon nuclei are 133.092 and 124.596 ppm, respectively. In methyl 9‐azido‐12Z‐octadecenoate (bis homoallylic), the carbon chemical shift of the ethylenic C‐12 and C‐13 carbon nuclei are 128.118 and 131.243 ppm, respectively.
Analysis of lipoxygenase kinetics by high‐performance liquid chromatography with a polymer column
Authors: Nuñez Alberto , Piazza George J.
Published: November 2006

Soybean lipoxygenase (LOX; EC 1.12.11.12) catalyzes the oxygenation of polyunsaturated fatty acids, acylglycerols and phosphoglycerols, producing a regio‐and enantiospecific hydroperoxide product. The goal of this work was to measure the relative rate of LOX‐catalyzed oxidation of mixtures of lipids containing linoleate, using high‐performance liquid chromatography (HPLC) and a light‐scattering detector (LSD). Previous literature sugested that reversed‐phase HPLC with silicabased columns could be used for the separation of individual fatty acids, acylglycerols, phosphoglycerides and their oxidation products. However, these columns produced ineffective separations of phosphoglycerides unless choline chloride and a strong base, such as KOH, are present in the mobile phase. Such modifiers precluded the use of the LSD. It was found that a reversed‐phase column based upon an organic polymer support, rather than on silica, was able to separate these mixtures with a ternary solvent gradient of methanol/water/acetonitrile without the need for the addition of modifiers. The oxidation time course of a mixture of linoleic acid, trilinolein and 1‐linoleoyl‐2‐stearoyl‐sn‐glycero‐3‐phosphocholine was followed using the developed HPLC method. The results showed that trilinolein and phosphatidylcholine reacted at one‐tenth the rate of linoleic acid. The diacylglycerol, 1,3‐dilinolein, was oxidized at a rate that was approximately 40% that of linoleic acid, with the formation of mono‐and dihydroperoxides as well as other unidentified products.
Effect ofd‐α‐tocopherol analogues on lipoxygenase‐dependent peroxidation of phospholipid‐bile salt micelles
Authors: Arai Hirofumi , Nagao Akihiko , Terao Junji , Suzuki Tetsuya , Takama Kozo
Published: November 2006

In order to know whether or not vitamin E acts as an effective antioxidant in lipoxygenase‐dependent peroxidation of phospholipids, the effect of vitamin E and vitamin E analogues, 2,2,5,7,8‐pentamethyl‐6‐hydrohychroman (PMC) and 6‐hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylic acid (Trolox C), was investigated in enzymatic lipid peroxidation of bile salt micelles of pig liver phosphatidylcholine (PC) using soybean lipoxygenase. 15‐Hydroperoxy‐5,8,11,13‐eicosatetraenoic acid was exlusively produced by the reaction with the PC molecular species containing arachidonic acid moiety, indicating that the hydroperoxidation of pig liver PC entirely progresses through the enzymatic reaction. PMC suppressed the accumulation of PC‐hydroperoxides (PC‐OOH) more efficiently than eitherd‐α‐tocopherol (α‐Toc) or Trolox C, and 50% inhibition concentration by PMC was close to that of quercetin, a known lipoxygenase inhibitor from natural origin. The antioxidant activity of PMC was also superior to that of either α‐Toc or Trolox C in ferrous ion‐induced nonenzymatic oxidation of PC micelles in the presence of a trace amount of PC‐OOH, although the radical‐scavenging activities of these compounds in solution were similar or comparable to one another. In conclusion, PMC is more effective than α‐Toc as an inhibitor of lipoxygenase reaction with phospholipids and of autoxidation in phospholipids. The phytyl chain of α‐Toc seems to be unfavorable for exerting an inhibitory effect on lipoxygenase reaction with phospholipid‐bile salt micelles.
Protective effect of estrogens and catecholestrogens against peroxidative membrane damagein vitro
Authors: Lacort Mercedes , Leal Ana M. , Liza Mariana , Martín César , Martínez Rosa , Ruiz‐Larrea M. Begoña
Published: November 2006

The antioxidant effects of natural estrogens (estrone E1; 17β‐estradiol), synthetic estrogens (17α‐ethynylestradiol, EE2; mestranol, MES; diethylstilbestrol, DES) and catechle‐strogens (2‐hydroxyestradiol; 4‐hydroxyestradiol 4‐OHE2) on lipid peroxidation induced by different means in rat liver microsomes were investigated. The extent of lipid peroxidation was determined by measuring thiobarbituric acid reactive substances. Prooxidants included Fe3+/ADP/reduced NADPH, Fe2+/ascorbate,tert‐butyl hydroperoxide (t‐BOOH) and 2,2′‐azobis (2‐amidinopropane) (AAPH). Estrogens and catecholestrogens decreased lipid peroxidation in all four systems tested. In the iron/ascorbate model it was shown that (i)‐OHE2 and DES had analogous patterns of inhibition, irrespective of the presence of NADPH or the functional integrity of the microsómes, and (ii) the antioxidant activities of E1, EE2 and MES were dependent on the assay conditions with the activity being markedley higher when estrogen metabolism was favored. When peroxidation was initiated by the peroxyl radical generator AAPH, the inhibitory effects observed were least pronounced. Our data also showed that, in each of the systems, all inhibitors displayed the same order of inhibitory potency with DES and catecholestrogens being the most potent antioxidants under all experimental conditions used. The present results confirm earlier findings and point toward a link between estrogen metabolism and estrogen antioxidant activity. The data also indicate that estrogens and catecholestrogens interact with the peroxidative process at different levels with their interactions with iron or the metal‐derived species being the most important modes of inhibition.
Effect of reproductive states on lipid mobilization and linoleic acid metabolism in mammary glands
Authors: Bandyopadhyay Gautam K. , Lee Le‐Young , Guzman Raphael C. , Nandi Satyabrata
Published: November 2006

Effects of pregnancy and lactation on lipid metabolism in mouse mammary fat pads and nonmammary adipose tissues have been studied. In order to address the question whether the influence of hormonal milieu on lipid metabolism in mammary epithelial cells during pregnancy and lactation is the same as in fat cells, we have studied the mobilization of lipids and metabolism of fatty acids in the intact mammary glands, parenchyma‐free mammary fat pads and in the perimetrial fat tissues of virgin, pregnant and lactating mice. Compared to parenchyma‐free mammary fat pads, the perimetrial adipose tissues accumulated 5‐fold higher levels of triglycerides during pregnancy. Mammary fat cells maintained overall lipid levels during pregnancy and lactation (16–20 μg/fat pad). In contrast, lactation depleted total lipid stores from 108 ± 5 to 24 ± 4.5 μg/fat pad in perimetrial fat pads. Results of comparative analysis of fatty acid composition of mammary fat pads, with and without epithelial tissue, from virgin and lactating mice showed stimulation of 18∶2ω6 metabolism leading to 130% increase in the ratio 20∶4ω6 to 18∶2ω6 in the epithelial compartment. Pregnancy and lactation resulted in the elevation of 20∶4ω6 levels probably due to a 4‐fold increase in Δ5 desaturase activity and a decrease in oxidative degradation of 18∶2ω6. These results suggest that, unlike other adipose tissues, the metabolic pathways in mammary fat cells are not dedicated to sequestration and accumulation of dietary lipids during pregnancy. Lactation favors mammary epithelial cell‐stimulated production of precursors of eicosanoids which are known to have agonist‐like effect on mammary epithelial cells.
1‐O‐alkyl‐2,3‐diacylglycerols in the skin surface lipids of the hairless mouse
Authors: Oku Hirosuke , Shudo Jyutaro , Mimura Kunio , Haratake Akinori , Nagata Junichi , Chinen Isao
Published: November 2006

A neutral lipid class was isolated by thin‐layer chromatography from the skin surface lipids of the hairless mouse. The fraction migrated faster than triglycerides and had a migration rate similar to that of diacyl alkanediols (diester wax). Upon deacylation, however, the long‐chain diols were identified as 1‐alkylglycerol ethers based on their chromatographic properties and on the mass spectra of their nicotinylidene derivatives. Thus, the skin lipid fraction was identified as 1‐O‐alkyl‐diacylglycerol. The alkyl moieties were all saturated and even‐numbered and ranged in chainlength from C16 to C22 with 1‐O‐hexadecylglycerol amounting to 34% of the total glycerol ether moieties. The fatty acids derived from this lipid fraction were mostly monoenoic with chainlengths ranging from C16 to C24. The major acyl component was eicosenoic acid (20∶1) representing 61% of the total fatty acids.
Analysis of triglycerides using atmospheric pressure chemical ionization mass spectrometry
Authors: Byrdwell Wm. Craig , Emken Edward A.
Published: November 2006

Atmospheric pressure chemical ionization (APCI) mass spectrometry was investigated as a new method for analysis of a mixture of triglycerides separated by reverse‐phase high‐performance liquid chromatography. A mixture of homogeneous (monoacid) triglyceride standards containing fatty acids with zero to three double bonds was analyzed to demonstrate the quality of mass spectra obtained by using the APCI interface. The mass spectra showed that minimal fragmentation occurs, resulting primarily in diglyceride [M−RCOO]+ ions and [M+1]+ protonated molecular ions. The degree of unsaturation within the acyl chains had a marked effect on the proportion of diglyceride ions vs. the [M+1]+ ions formed in the APCI source. The mass spectra of triglycerides containing fatty acids with two or three double bonds showed predominantly protonated triglyceride ions, with diglyceride peaks representing 13 to 25% of the base peak. The triglycerides containing singly unsaturated fatty acids gave diglyceride ions as the base peak, and [M+1]+ ions with an intensity 20 to 28% that of the base peak. Only diglyceride ions were observable in the spectra of triglycerides containing saturated fatty acids.
Effect of growth temperature on the positional distribution of eicosapentaenoic acid andrans hexadecenoic acid in the phospholipids of aVibrio species of bacterium
Authors: Henderson R. J. , Millar R. M. , Sargent J. R.
Published: November 2006

Phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) were isolated from aVibrio species of bacterium, known to produce eicosapentaenoic acid (20∶5n−3) andtrans‐hexadecenoic acid (16∶1n−7), and subjected to phospholipase A2 degradation to determine the positional distribution of component fatty acids. At the two growth temperatures studied (20 and 5°C), both 20∶5n−3 andtrans 16∶1 n−7 were located mainly at positionsn−2 in PE. Increases in the proportions of 20∶5n−3 andtrans 16∶1n−7 in positionsn−2 with decreasing growth temperature were balanced mainly by decreases in the level ofiso‐15∶0. In PG,trans 16∶1n−7 was located predominantly in positionsn‐1, although the difference between the two positions was not as great as in PE. Eicosapentaenoic acid was preferentially located in positionsn‐2 of PG, particularly at 5°C when it comprised 29.9% of the total fatty acids in this position. It is concluded thattrans 16∶1n−7/20∶5n−3 is not a major molecular species of phospholipid in this species ofVibrio and that changes in the levels of molecular species of PE containingiso‐15∶0 may feature in thermal acclimation.
β‐Carotene transport in human lipoproteins. Comparisons with a‐tocopherol
Authors: Traber Maret G. , Diamond Seth R. , Lane Jerome C. , Brody Rachel I. , Kayden Herbert J.
Published: November 2006

The purpose of this study was to investigate the temporal relationships of the transport of β‐carotene in human lipoproteins. We administered 60 mg β‐carotene with breakfast to nine fasting subjects, then blood samples were collected at intervals of up to 75 h, lipoproteins were isolated, and β‐carotene was quantitated. β‐Carotene concentrations in chylomicrons and very low density lipoproteins (VLDL) peaked at 6 and 9 h, respectively. Nonetheless, at all time points the majority of plasma β‐carotene was contained in low density lipoproteins (LDL), while high density lipoproteins (HDL) carried a smaller portion (at 24 h, 73±8% in LDL as compared with 23±5% in HDL). In three subjects, transport of β‐carotene was compred with the results of earlier studies on the transport of stereoisomers of α‐tocopherol. Unlike plasmaRRR‐α‐tocopherol concentrations, which are maintained by the preferential incorporation ofRRR‐α‐tocopherol into VLDL by the liver, β‐carotene increased and decreased in VLDL similarly toSRR‐α‐tocopherol, a stereoisomer whose concentrations are not maintained in plasma. In conclusion, β‐carotene is primarily transported in the plasma in LDL, but its incorporation by the liver into lipoproteins does not appear to be enhanced.
Upregulation of low density lipoprotein receptor activity by tumor necrosis factor, a process independent of tumor necrosis factor‐induced lipid synthesis and secretion
Authors: Liao Wei , Florén Claes‐Henrik
Published: November 2006

It has been shown that tumor necrosis factor (TNF) rapidly upregulates expression of the low density lipoprotein (LDL) receptors on Hep G2 cells and acutely stimulates hepatic lipid synthesis and secretionin vivo. It may thus be possible that TNF‐induced expression of LDL receptors is secondary to a decrease in cellular cholesterol content caused by TNF‐stimulated lipid secretion. In order to know whether TNF upregulates LDL receptors by depletion of the cellular cholesterol content, the present experiments were designed to study the temporal relationship between TNF‐stimulated expression of LDL receptor activity and TNF‐induced changes in lipid synthesis and secretion in anin vitro setting by using Hep G2 cells (a highly differentiated human hepatoma cell line) as a hepatocyte model. Hep G2 cells were incubated with TNF (usually 2.5 nmol/L) for certain periods, and LDL receptor activity was evaluated by measuring [125I]LDL binding at 4°C; lipid synthesis and secretion were assayed by measuring [3H]glycerol incorporation into triglycerides and phospholipids as well as [14C]acetate incorporation into cholesterol. We found that a 30‐h exposure of the cells to TNF was needed for the effect of TNF to be seen on lipid synthesis and secretion as measured by incorporation of [3H]glycerol into triglycerides and phospholipids, whereas TNF rapidly (in several hours) upregulated LDL receptor activity. TNF stimulated triglyceride synthesis, but did not stimulate phospholipid synthesis. On the other hand, TNF stimulated phospholipid secretion, but did not stimulate triglyceride secretion. Exposure of the cells to TNF for 16 or 24 h neither decreased cholesterol synthesis nor stimulated cholesterol secretion as measured by [14C]acetate incorporation into cholesterol. Upregulation of LDL receptor activity through inhibition of cellular cholesterol synthesis with compactin (a competitive inhibitor of the 3‐hydroxyl‐3‐methylglutaryl‐CoA reductase) was augmented by TNF, whereas downregulation of LDL receptor activity through stimulation of cellular cholesterol synthesis with mevalonolactone almost completely blocked the upregulatory effect of TNF. In conclusion, TNF‐stimulated expression of LDL receptor activity is not secondary to a depletion of cellular cholesterol content through TNF‐stimulated lipid secretion or inhibition of cholesterol synthesis.
Linoleic acid uptake by isolated enterocytes: Influence of α‐linolenic acid on absorption
Authors: Goré Jacques , Hoinard Claude , Couet Charles
Published: November 2006

In a previous study we showed that intestinal uptake of α‐linolenic acid (18∶3n−3) was carrier‐mediated and we suggested that a plasma membrane fatty acid protein was involved in the transport of long‐chain fatty acids. To further test this hypothesis, the mechanism of linoleic acid (18∶2n−6) uptake by isolated intestinal cells was examined using a rapid filtration method and 20 mM sodium taurocholate as solubilizing agent. Under these experimental conditions transport of [1‐14C]linoleic acid monomers in the concentration range of 2 to 2220 nM was saturable with a Vm of 5.1±0.6 nmol/mg protein/min and a Km of 183±7 nM. Experiments carried out in the presence of metabolic inhibitors, such as 2,4‐dinitrophenol and antimycin A, suggested that an active, carriermediated mechanism was involved in the intestinal uptake of this essential fatty acid. The addition of excess unlabeled linoleic acid to the incubation medium led to a 89% decrease in the uptake of [1‐14C]linoleic acid, whiled‐glucose did not compete for transport into the cell. Other long‐chain polyunsaturated fatty acids added to the incubation mixture inhibited linoleic acid uptake by more than 80%. The presence of α‐linolenic acid (18∶3n−3) in the incubation medium caused the competitive inhibition (Ki=353 nM) of linoleic acid uptake. The data are compatible with the hypothesis that intestinal uptake of both linoleic, and α‐linolenic acid is mediated by a membrane carrier common to long‐chain fatty acids.
Occurrence of 5c,8c,11c,15t‐eicosatetraenoic acid and other unusual polyunsaturated fatty acids in rats fed partially hydrogenated calola oil
Authors: Ratnayake W. M. N. , Chen Z. Y. , Pelletier G. , Weber D.
Published: November 2006

Uncommoncis andtrans fatty acids can be desaturated and elongated to produce unusual C18 and C20 polyunsaturated fatty acids in animal tissues. In the present study we examined the formation of such metabolites derived fromcis andtrans isomers of oleic and linoleic acids of partially hydrogenated vegetable oil origin in rats. For two months, aduut male rats were fed a partially hydrogenated canola oil diet containing moderately high levels oftrans fatty acids (9.6 energy%) and an adequate level of linoleic acid (1.46 energy%). Analysis of the phospholipid (PL) fatty acids of liver, heart, serum and brain showed no new C18 polyunsaturated fatty acids, except for those uncommon 18∶2 isomers originating from the diet. However, minor levels (each <0.3% PL fatty acids) of six unusual C20 polyunsaturated fatty acids were detected in the tissues examined, except in brain PL. Identification of their structures indicated that the dietary 9c,13t−18∶2 isomer, which is the majortrans polyunsaturated fatty acid in partially hydrogenated vegetable oils, was desaturated and elongated to 5c,8c,11c,15t−20∶4, possibly by the same pathway that is operative for linoleic acid. Furthermore, dietary 12c−18∶1 was converted to 8c,14c−20∶2 and 5c,8c,14c−20∶3; dietary 9c,12t−18∶2 metabolized to 11c,14t−20∶2 and 5c,8c,11c14t−20∶4, and dietary 9t,12c to 11t,14c−20∶2. These results suggested that of all the possible isomers of oleic and linoleic acids in partially hydrogenated vegetable oils, 12c−18∶1, 9c,13t−18∶2, 9c,12t−18∶2 and 9t,12c−18∶2 are the preferred substrates for desaturation and elongation in rats. However, their conversions to C20 metabolites were not as efficient as that of oleic or linoleic acids.