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Characterization of a lipid-rich fraction synthesized by Streptomyces avermitilis.

Isolation of the macrocyclic lactone parasiticide avermectin and other closely related natural products produced by Streptomyces avermitilis also yields a lipid-rich fraction. The latter has been characterized by techniques based on gas-liquid chromatography (GLC) and mass spectrometry (MS). Initial examination of the lipid-rich fraction by direct probe electron-impact (EI) MS and packed-column GLC showed that it consists primarily of a mixture of triglycerides possessing C14-C17 acyl groups. Further examination of this fraction by capillary column GLC-MS demonstrated that it contains low levels of C15-C17 free fatty acids, squalene and diglycerides and, as the major components, at least ten mixed acyl triglycerides (total number of acyl carbon atoms ranging from 43 to 50). Prominent among the triglycerides were a C15-C15-C16 species, a C15-C16-C16 species and a C15-C16-C17 species. Capillary-column GLC and GLC-MS of the fatty acid methyl esters resulting from transesterification demonstrated that the major triglyceride acyl groups are anteiso-C15 (12-methyltetradecanoyl), iso-C16 (14-methylpentadecanoyl), n-C16 (hexa-decanoyl) and anteiso-C17 (14-methylhexadecanoyl). Lower levels of the methyl esters of the following fatty acids were observed: iso-C14 (12-methyltridecanoic), n-C14 (tetradecanoic), iso-C15 (13-methyltetradecanoic), n-C15 (pentadecanoic), iso-C17 (15-methylhexadecanoic) and n-C17 (heptadecanoic). Little evidence was seen for either unsaturated acyl groups or acyl groups of less than 13 or more than 18 carbon atoms. Desorption chemical ionization MS (ammonia reagent gas) analysis confirmed the nature of the lipid-rich fraction, and is an attractive one-step approach for determining the molecular weights and distribution of triglycerides in a mixture.

Chemical Phenomena↗

Gas-liquid chromatography-mass spectrometry of phospholipid mixtures after enzymic hydrolysis.

Gas-liquid chromatography-mass spectrometry (GLC-MS), employing an open-tubular Silanox-type glass column, has been applied to the products of phospholipase C hydrolysis of natural and synthetic phospholipid mixtures. The materials studied were egg lysolecithin, synthetic L-alpha-l-stearoyl-2-oleoyl lecithin, bovine brain sphingomyelin, and phospholipids derived from human arterial tissue. l-Monoglycerides and ceramides were analysed as methaneboronates, and 1,2-diglycerides as trimethylsilyl ethers. The results indicate the potential value of open-tubular GLC-MS in a rapid procedure for the concurrent analysis of the major classes or polar lipids after enzymic dephosphorylation.

Chromatography, Gas↗

Effect of isoproterenol on the metabolism of myocardial fatty acids.

The effect of beta-adrenergic stimulation on myocardial fatty acid metabolism was studied in the anesthetized open-chest dog. Isoproterenol (1 microgram/kg/min) was infused; and samples of arterial blood and of left ventricular wall were taken at the 5th min for the determination of the following variables: in arterial blood: lactate and serial free fatty acids (FFA); in myocardial tissue: creatine phosphate (CP), ATP, lactate, carnitine, acylcarnitine, glycerol, and the fatty acid content of each of phospholipids (PL), cholesterol esters (CE), triglycerides (TG), diglycerides (DG), monoglycerides (MG), and FFA. A capillary gas chromatography was used for fatty acid assay. Isoproterenol decreased the content of creatine phosphate but not of ATP. Lactate increased in both arterial blood and myocardial tissue. The five-fold increase in arterial FFA (P less than 0.001) was accompanied with a significant increase in FFA serum/tissue ratio. Free carnitine decreased and acylcarnitine increased. Triglycerides content (which is expressed in terms of its total fatty acid content) was considerably reduced by beta-stimulation in comparison with control group (2694 +/- 689 vs. 7518 +/- 833 nmol/g wet wt, P less than 0.001), and tissue glycerol increased (P less than 0.01). The decrease in total MG content was significant, but not that of DG, nor that of CE. Total PL content did not change. The most marked individual changes (except in PL) were observed on monounsaturated fatty acids, 18: 1 omega 9, 18: 1 omega 7, and 16: 1 cis. The significant changes of monounsaturated/saturated and of 16:1 cis/16:1 trans ratios, in arterial FFA and tissue TG, suggested modifications in the distribution of fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

The role of fatty acid composition and positional distribution in fat absorption in infants.

Fat digestion and absorption in the infant is a multistep process. An initial gastric phase of lipolysis generates modest amounts of diglycerides, monoglycerides, and free fatty acids. These initial digestion products, as well as bile salts, are required for optimal activity of the intestinal phase of lipolysis. Colipase-dependent pancreatic lipase catalyzes the intraduodenal phase of triglyceride digestion in formula-fed infants; in breast-fed infants this process is also mediated by bile salt-stimulated lipase. Triglyceride fatty acid positional distribution may modulate the efficiency of nutrient absorption. Human milk contains palmitic acid (C16:0) primarily in the sn-2 position; infant formula fat blends contain palmitic acid predominantly in the sn-1 and sn-3 positions. Because pancreatic lipase selectively hydrolyzes triglycerides at the sn-1 and sn-3 positions, free fatty acids and 2-monoglycerides are produced. Free palmitic acid, but not 2-monopalmitin (which is efficiently absorbed), may be lost as a calcium-fatty acid soap in the feces. As a result, many infant formulas contain substantial levels of well-absorbed saturated fatty acids of shorter chain lengths (e.g., C12:0) in place of palmitic acid. Means of increasing the proportion of 2-palmitic acid in infant formula may make possible fat blends closer to that of human milk with acceptable absorption characteristics.

Animals↗

2-Chloroadenosine reverses hyperglycemia-induced inhibition of phosphoinositide synthesis in cultured human retinal pigment epithelial cells and prevents reduced nerve conduction velocity in diabetic rats.

The effect of the adenosine (AD) analog 2-chloroadenosine (C-AD) on glucose-induced inhibition of phosphoinositide synthesis was studied in human retinal pigment epithelial (RPE) cells by monitoring the level of the phosphatidylinositol (PI) synthase substrate, cytidine diphosphate diglyceride (CDP-DG). In high-aldose reductase (AR)-expressing RPE 91 cells, C-AD decreased CDP-DG at 5 mmol/L glucose and reversed the increase by 20 mmol/L glucose. AD deaminase (ADA), which inactivates endogenously released AD, potentiated the hyperglycemia-induced increase in CDP-DG. Theophylline, an AD-A1 and AD-A2 receptor antagonist, caused an increase in CDP-DG at 20 mmol/L glucose. C-AD did not alter CDP-DG in low-AR-expressing RPE 45 cells, but did decrease CDP-DG after cells were conditioned in 300 mmol/L glucose for 1 week (which induces AR). The mechanism by which AD regulates PI synthase in cells with high AR activity is unknown, but it is independent of Gi or Gs proteins, adenylate cyclase and phospholipase C (PLC) activation, myo-inositol (MI) uptake, or MI efflux. Administration of C-AD to streptozotocin-induced diabetic rats prevented the slowing of motor nerve conduction velocity (MNCV). Thus, AD derivatives, which reverse a glucose-induced deficit in phosphoinositide metabolism, might serve as a useful pharmacological tool to intervene in hyperglycemia-induced diabetic complications.

2-Chloroadenosine↗

Altered cord serum lipid levels associated with small for gestational age infants.

OBJECTIVE: To determine whether small for gestational age (SGA) infants show changes in lipid metabolism that could distinguish growth-restricted subpopulations. METHODS: Sera from the arterial cord blood from 38 SGA infants were analyzed for apolipoprotein A-I level, total lipid content, and distribution of those lipids as triglycerides, diglycerides, free fatty acids, and phospholipids. Comparisons were made between appropriate for gestational age (AGA) controls (n = 25), SGA infants with a ponderal index below the tenth percentile (SGA I, n = 20), and SGA infants with a ponderal index above the tenth percentile (SGA II, n = 18). RESULTS: Total cord serum lipid content was markedly decreased in all SGA infants compared with AGA infants (2.8 times lower). Although SGA infants showed total lipid concentration decreases, SGA I and SGA II infants showed distinct characteristics. Infants in the SGA I group had higher triglyceride levels (1.8 times higher) and lower free fatty acid levels (1.4 times lower), compared with AGA infants (P < .001). The lipid subclass distribution in SGA II infants was not significantly different from that in AGA infants, with the exception of an increase in triglyceride concentrations (1.3 times higher). Although the 22-kD placenta-derived apolipoprotein A-I was similar in all groups, the level of fetal liver-derived 28-kD apolipoprotein A-I was 6.5 times lower in SGA I infants than in AGA or SGA II infants (P < .001). CONCLUSION: The SGA I infants appeared to have impaired utilization of circulating triglycerides, consistent with peripheral adipose depletion. Diminished fetus-derived apolipoprotein A-I levels with normal levels of placenta-derived apolipoprotein A-I levels might indicate a defect in the production or secretion of apolipoproteins associated with growth restriction.

Fetal Blood↗

Stimulatory effect of progesterone and 5 beta-progesterone on lipid synthesis in hamster flank organs.

In order to study lipid synthesis in female hamster flank organs during the estrous cycle, glands were obtained from different animals during different phases of the cycle. Lipid metabolism of [U-14C]glucose was studied under in vitro conditions. The radioactive lipids formed, were extracted from the glands and quantified. An aliquot of the extract was submitted to TLC to isolate and identify the lipids formed. Lipid synthesis in the glands increased significantly during estrous compared with the diestrous phase, suggesting the influence of sex hormones on lipid metabolism in flank organs. The radioactive lipids isolated and identified were: phospholipids, cholesterol, glycerides, waxes, and cholesterol esters. The percentages of conversation from [U-14C]glucose to cholesterol esters were minor in extracts from glands of diestrous animals as compared to estrous and proestrous animals, whereas glycerides and waxes increased significantly in metestrous and diestrous animals. To verify and evaluate the role of estrogen and progesterone in lipid synthesis, three different groups of gonadectomized female hamsters were injected daily with estradiol, progesterone, and 5 alpha-progesterone. After treatments, glands were incubated with [U-14C]glucose to determine the incorporation of radioactive glucose into lipids under culture conditions. The radioactive lipids synthesized were subsequently extracted and identified. The results showed that estrogen had no effect on in vitro [U-14C]glucose incorporation into lipids by flank organs, compared to the vehicle, whereas progesterone and 5 alpha-progesterone increased radioactive lipid synthesis by the glands significantly (p < 0.05). The radioactive lipids isolated and identified from extracts of gonadectomized female hamster flank organs were: phospholipids, cholesterol, triglycerides, waxes, and cholesterol esters. Gonadectomy increased phospholipid synthesis and decreased that of monoglycerides, diglycerides, and waxes. The percentages of conversion from [U-14C]glucose to cholesterol esters were higher after progesterone and 5 alpha-progesterone treatments than in vehicle and estrogen-treatments. Our data indicate that progesterone and 5 alpha-progesterone-treatments increased the in vitro [U-14C]glucose incorporation into lipids in flank organs from gonadectomized female hamsters. Furthermore, the major lipid synthesized by glands under these stimuli were cholesterol fatty acids. Thus, progesterone and 5 alpha-progesterone alter the consistency of sebum from gonadectomized female glands.

Animals↗

Progesterone induces meiotic division in the amphibian oocyte by releasing lipid second messengers from the plasma membrane.

Meiosis in the amphibian oocyte is normally initiated by gonadotropins, which stimulate follicle cells to secret progesterone. The progesterone-induced G2/M transition in the amphibian oocyte was the first well-defined example of a steroid effect at the plasma membrane, since it could be shown that exogenous, but not injected, progesterone induced meiosis and that many of the progesterone-induced changes associated with meiosis occurred in enucleated oocytes. We find that [3H]progesterone binding to isolated plasma membranes of Rana pipiens oocytes is saturable, specific and temperature-dependent. Photoaffinity labeling with the synthetic progestin [3H]R5020 followed by gel electrophoresis demonstrated progestin binding to both 80 and 110 kDa proteins in the oocyte cytosol, whereas only the 110 kDa R5020 binding protein was present in the oocyte plasma membrane. We have shown that progesterone acts at Rana oocyte plasma membrane receptors within seconds to release a cascade of lipid messengers. Membrane-receptor binding causes the successive activation of: 1) N-methyltransferases, which convert phosphatidylethanolamine to phosphatidylcholine (PC); 2) an exchange reaction between PC and ceramide to form sphingomyelin (SM) and 1,2-diacylglycerol (DAG); 3) phospholipase D/phosphatidate phosphohydrolase, releasing a second DAG transient; and 4) phosphatidylinositol-specific phospholipase C, generating inositol trisphosphate and a third DAG transient. Within minutes, diglyceride kinase converts newly formed DAG species to phosphatidic acid, turning off the successive DAG signals. A transient fall (0-30 s) in intracellular ceramide is followed (within 1-2 min) by a sustained rise in intracellular ceramide lasting 3-4 h. This ceramide may be significant in later cyclin-dependent steps. We conclude that the initial action of progesterone at its plasma membrane receptor triggers a series of enzyme activations that modify the membrane and release multiple DAG species.

Animals↗

Occurrence of the toxin dehydroabietic acid in Salmonella typhimurium.

Many strains of Salmonella typhimurium studied in our lab demonstrated marked differences in the pathogenicity for guinea pig, chicken and Hela cells. As a result, a pathogenic strain of S. typhimurium, strain 9SR2, was evaluated for lipophilic components that may be associated with virulence using gas chromatography/mass spectrometry. The hydroxylated fatty acids 2-hydroxytetradecanoic acid (2-OH-14:0) and 3-hydroxytetradecanoic acid (3-OH-14:0) often present in lipid A, a potent endotoxin, were observed as their methyl esters. The cyclic fatty acids methylene-hexadecanoic acid (C17delta) and methyleneoctadecanoic acid (C19delta) also were detected. The nephrotoxic and neurotoxic diterpenoid resin acid, dehydroabietic acid, was observed for the first time from S. typhimurium in both the total lipid and diglyceride fractions and determined as its methyl ester at m/z 314.2246. Due to its previously established toxicity, dehydroabietic acid may be a factor associated with virulence of S. typhimurium.

Abietanes↗

Fetal surfactant as a source of arachidonate in human amniotic fluid.

The factors responsible for the onset of labor in women are not well understood but it is clear that parturition is associated with increased production of prostanoids and release of arachidonic acid by intrauterine tissues. Pulmonary surfactant is secreted from the fetal lung into the amniotic fluid where its concentration increases toward term. In this paper we have shown that the ability of fetal surfactant to stimulate prostaglandin production by amnion cells is greatly enhanced by pre-incubating surfactant with amniotic fluid. This is due to the release of fatty acids, including arachidonate, from the lipids of fetal surfactant by the sequential action of phospholipase C and diglyceride lipase. Thus, in addition to providing the amnion with a source of arachidonate derived from the intracellular transfer of arachidonate from surfactant phosphatidylcholine to phosphatidylethanolamine and phosphatidylinositol in amnion cells, fetal surfactant also contributes to the pool of free arachidonate in amniotic fluid.

Amniotic Fluid↗

Integration of phospholipid and sterol metabolism in mammalian cells.

The lethal consequences of imbalances in lipid and sterol metabolism in human diseases such as atherosclerosis and lipid storage disorders underscores our need to know how cholesterol, phospholipid and sphingolipid metabolism is integrated. Accumulation and abnormal localization of lipids and sterol affects cellular function not only by perturbing membrane activity but also by increasing production of bioactive lipids derived from cholesterol, phospholipids and sphingolipids. For example in the NPC mouse model, accumulation of intracellular cholesterol and sphingomyelin is accompanied by increased sphingosine [187], a potent regular of protein kinase C and cell proliferation [152]. Oxidized LDL has an important role in the pathology of atherosclerosis by promoting foam cell formation and cytotoxicity [65]. 7-Hydroxycholesterol and 7-ketocholesterol are involved in many aspects of oxidized LDL activity including initiation of apoptosis in a number of cell types [188, 189] and enhancing cholesterol accumulation by inhibiting efflux [190]. Oxysterols formed intracellularly or from oxidized lipoproteins could have an important role in regulating lipid metabolism in the foam cell. Bioactive metabolites of phospholipids, such as diglyceride, phosphatidic acid and lysolipids, could also increase in circumstances of elevated deposition and have profound and varied effects on cell physiology. In addition to elucidating mechanisms for integration of lipid metabolism, we should determine when these responses go awry and assess the influence of bioactive compounds formed under these circumstances on cell viability and growth.

Animals↗

The compound DATEM inhibits respiratory syncytial virus fusion activity with epithelial cells.

The effect of diacetyltartaric acid esters of mono and diglycerides (DATEM) on fusion of respiratory syncytial virus (RSV) with HEp-2 cells was studied using the R18 fluorescence dequenching fusion assay. At DATEM concentrations less than 2.0 microg/ml, the inhibition of fusion increased with the concentration of DATEM. At 2 microg/ml of DATEM, the fusion was suppressed by 80-90%. Studies examining possible mechanism of fusion-inhibition indicated that DATEM was likely adsorbed onto lipid membranes of both viral envelope and target cell membranes. Quantitative measurements of DATEM adsorption onto membranes were also performed using lipid monolayers and vesicles. The surface pressure of lipid monolayer formed at the air/aqueous interface increased as the concentration of DATEM in the monolayer subphase increased, suggesting that DATEM was inserted into the monolayer. As the concentration of DATEM in vesicle suspensions increased, electrophoretic mobility of initially uncharged lipid vesicles also increased, reflective of increased negative charge at vesicle surfaces. These results strongly suggest that the insertion of DATEM onto membranes inhibited viral fusion. DATEM may prove to be effective in limiting the infectivity of RSV by interference with the fusion of the viral envelope with target cell membranes.

Adsorption↗

Studies on ether-phospholipids of vascular smooth muscle cells. Identification of a rapid Ca(2+)-dependent hydrolysis of alkyl-phosphatidylethanolamine promoted by endothelin-l.

We have investigated the metabolism of 1-O-[3H]octadecyl-sn-glycero-3-phosphocholine ([3H]lyso PAF) and [3H] myristic acid in secondary cultures of aortic smooth muscle cells (SMC) to characterize the origin of second messengers generated upon stimulation with endothelin-l (ET-l). When cells were labelled with [3H]lyso PAF, we observed a transfer of the label from phosphatidylcholine (PC) to phosphatidylethanolamine (PE) In contrast, incubation with [3H]lyso PAF labelled mainly alkyl-subclasses while [3H]myristate was associated with diacyl-subclasses. Using these specific labelling procedures, we have shown that ET-l induced a strong hydrolysis of PE. This hydrolysis was specific for alkyl-PE with a maximum after 5 s of stimulation. We have also observed an extracellular Ca(2+)-dependent increase in diglyceride (DG), phosphatidic acid (PA) and mainly triglyceride (TG) concomitant to alkyl-PE hydrolysis. Thus, alkyl-DG generated from alkyl-PE appears to be a major product in ET-l stimulation of SMC. These results suggest a new level of complexity in the signal transduction cascade involving a specificity for phospholipid subclasses.

Animals↗

Efficient lipase catalysed production of a lubricant and surfactant formulation using a continuous solvent-free process.

The transesterification of sunflower oil with a high oleic acid residue content (typically 83.5%) with butanol-1 by immobilised Lipozyme was carried out in a solvent free system and in a continuous way. During the first 6 h of reactor operation, a transition phase was observed, in which the main products were butyl ester and glycerol. This latter being insoluble in the reaction mixture, it is adsorbed onto the enzyme support thus leading to a decrease in enzyme performance. Step by step, less and less glycerol is produced and finally when glycerol is no longer produced a steady state is attained. The product composition is a mixture of butyl ester (65 molar%), monoglyceride (26 molar%), diglyceride (6 molar%) and residual triglyceride (3 molar%). This mixture has interesting lubricant and surfactant properties. The reactor was maintained without any loss in activity for a period of 3 months. This result is very different to that obtained using an organic solvent (n-hexane) which leads to a total loss of enzyme activity within a few hours.

Bioreactors↗

In vitro models for the identification and characterization of tumor-promoting and protective factors for colon carcinogenesis.

The present review aims to give a short overview of the existing cell culture models and their characteristics. While both the induction and the prevention of tumors can only be unequivocally demonstrated in vivo, in vitro models of colorectal cell growth are essential tools for the identification of candidate compounds and for the analysis of underlying mechanisms. Unfortunately normal and premalignant colorectal epithelial cells that represent the target cells of tumor promoters like desoxycholic acid or 1,2-diglyceride and could be used to investigate their cell biological effects are difficult to obtain. Cell line studies have mostly used carcinoma cell lines or rat small intestinal epithelial cells. Some normal immortalized cell lines have been established but are not widely used, and premalignant adenoma cell lines are extremely rare. However, those that do exist are useful both in mechanistic studies of cell growth and for the analysis of tumor promoters and chemoprotectors of colorectal carcinogenesis.

Adenoma↗

Mechanical properties of model membranes studied from shape transformations of giant vesicles.

Membrane deformations occur frequently in cell functioning. From the physical point of view, the understanding of such shape changes requires the introduction of mechanical parameters like bending elasticity. In this article it is shown how this physical property can be obtained from the analysis of small or large shape transformations from giant vesicles. Then it is demonstrated that the bending modulus is strongly dependent on the membrane composition and environmental conditions. This is the case for one-component bilayers (dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine and stearoyloleoyl-phosphatidylcholine (SOPC) and for two-component lipid mixtures (DMPC/cholesterol, DLPC/dilauroylphosphatidic acid). Further it is shown that the bending elasticity of natural lipid extracts (egg phosphatidylcholine, digalactosyl diglyceride and red blood cell lipid extracts) is generally smaller than that of comparable synthetic model membranes. The role of transmembrane proteins is examined by measuring the bending elasticity of SOPC/gramicidin mixtures. Finally, larger scale shape transformations of giant vesicles under an alternative electric field are discussed.

Elasticity↗

Phosphatidic and lysophosphatidic acid production in phospholipase C-and thrombin-treated platelets. Possible involvement of a platelet lipase.

Incubation of 32P-labelled platelets with Clostridium welchii phospholipase C greatly stimulates 32P-incorporation into phosphatidic and lysophosphatidic acids. A net synthesis is demonstrated for both phospholipids, which exhibit identical specific radioactivities. Phosphatidic acid production roughly parallels the phospholipase C-induced aggregation, whereas lysophosphatidic acid appears secondarily during cell lysis. The same qualitative variations are observed during thrombin-induced aggregation. At the physiological pH used throughout the incubations, platelets display no phospholipase A activity towards phosphatidic acid, whereas diglycerides are deacylated by platelet lysates. On the basis of these findings, a mechanism for phosphatidic and lysophosphatidic acid production is proposed, involving a phosphorylation of the di- and monoglycerides formed upon phospholipase C and lipase action. The possible role of such a pathway in regulating arachidonic acid release from phospholipids during platelet activation is discussed.

Animals↗

Characterisation and quantification of medium chain and long chain triglycerides and their in vitro digestion products, by HPTLC coupled with in situ densitometric analysis.

The development of new and simple high performance thin layer chromatography (HPTLC) assays for the quantification of medium chain triglycerides (MCT, tricaprylin) and long chain triglycerides (LCT, triolein) and their lipolytic products, bile salts (BS) and phospholipids (PL) are described. Different classes of lipids (PL, BS, fatty acids, monoglycerides, diglycerides, and triglycerides) were separated on a single silica gel 60 HPTLC plate by Automated Multiple Development (AMD) methods using a Camag AMD 2. Post-chromatographic staining of long chain lipids (triolein, diolein, monoolein, and oleic acid), PL and BS with a solution of copper sulphate-phosphoric acid and medium chain lipids (tricaprylin, dicaprylin, monocaprylin, and caprylic acid) with a solution of ammonium molybdate-perchloric acid allowed visualisation of the lipids. Lipids were quantified by in situ spectrodensitometric measurements using a Camag TLC scanner 3. The intra- and inter-assay accuracy was between 83 and 115% and the assay was precise to within a CV of less than 20% over a range of 0.1-1 and 5-50 microg for long chain lipids and medium chain lipids, respectively. The methods have been employed to study the kinetics of triolein and tricaprylin lipolysis in an in vitro lipid digestion model commonly used to assess the digestibility of novel oral lipid-based formulations.

Chromatography, Thin Layer↗