Quantitative determination of acyl chain composition of subnanomole amounts of cellular long-chain acyl-coenzyme A esters

A procedure for the quantitative determination of the acyl chain composition of cellular long-chain acyl-CoA esters in subnanomole amounts is described. The abundant cellular lipids of samples are removed by extraction with organic solvents, and the proteins are precipitated from the aqueous phase by the addition of acetonitrile. The CoA thiolesters are adsorbed on neutral aluminum oxide and reduced with sodium borohydride to the corresponding alcohols that are then converted to t-butyldimethylsilyl ethers and analyzed quantitatively by gas chromatography. Saturated and unsaturated acyl chains behaved similarly throughout the procedure, and the common lipid esters do not interfere with the analysis of the CoA esters in the final assay procedure described. This simple and relatively rapid method is suitable for analyzing a large number of samples at a time.     

Changes in the physico-chemical characteristics of mouse liver lipids after administration of a mixture of unsaturated fatty acids

The effects of a mixture of unsaturated fatty acids (UFA) on changes in the lipid oxidation substrate, a system of natural antioxidants and the functioning of a system controlling lipid peroxidation were studied. It was shown that arachidonate induces the incorporation of fatty acids into individual lipid fractions and the dilution of lipids by UFA esters, as a result of which the relative amount of natural antioxidants diminishes. Under these conditions, the oxidative capacity of lipids decreases, which manifests itself in a reduction of the rate of natural antioxidants utilization and an increase in their number. It was shown that purposeful modification of the lipid substrate does not interfere with the interaction between individual parameters of the system controlling lipid peroxidation.     

High concentrations of magnesium modulate vascular endothelial cell behaviour in vitro

Magnesium supplementation has been reported to prevent cardiovascular diseases through the decrease of plasma lipids and to improve endothelial function in patients with coronary artery disease. In the present work, we evaluated whether high magnesium concentrations can directly affect the function of cultured endothelial cells, which play a crucial role in maintaining the functional integrity of the vascular wall. We cultured human umbilical vein endothelial cells for various times in media containing different concentration of magnesium (range 2 to 10 mM) and compared them to the corresponding controls (1 mM Mg). High Mg concentrations stimulated endothelial proliferation, enhanced the motogenic response to angiogenic factors and attenuated the response to lipopolysaccharide (LPS). In addition, we demonstrate that high concentrations of magnesium did not modulate the levels of plasminogen activator inhibitor-1, but enhanced the synthesis of nitric oxide, in part through the up-regulation of endothelial nitric oxide synthase. Our results demonstrate a direct role of magnesium in maintaining endothelial function. We therefore anticipate that magnesium may have a protective effect against atherosclerosis and could play a role in promoting the growth of collateral vessels in chronic ischemia. Moreover, because it induces the synthesis of nitric oxide, this cation could be a helpful tool in hypertension as well as in preventing thrombosis.     

Same-day batch measurement of glycine betaine, carnitine, and other betaines in biological material.

Glycine betaine, carnitine, carnitine esters, butyrobetaine, and proline betaine (stachydrine) concentrations in biological materials can be reliably measured in 100-microliters samples, with a detection limit below 1 mumol/liter. The procedure is suitable for batches of more than 30 specimens and it is possible to obtain a single result within 2 h. The betaines are extracted into an acetonitrile:methanol mixture, dried with anhydrous disodium hydrogen phosphate containing argentous oxide. The 4-bromophenacyl ester derivatives are formed using 4-bromophenacyl triflate as reagent, in the presence of solid magnesium oxide as base. The derivatives are separated by high-performance chromatography on a silica column, in a mixed partition and ion-exchange mode.     

Distribution of arachidonic and eicosapentaenoic acids in the lipids of mosses.

Lipid classes from four species of mosses, Mnium cuspidatum, and Mnium medium from Minnesota, and Hylocomium splendens and Pleurozium schreberi from Alaska, were analyzed. The total lipids of all species contained 30-40% arachidonic and eicosapentaenoic acids. However, the lipids from the Alaskan mosses contained about 75% neutral lipids (triacylglycerols, steryl esters and wax esters) whereas the lipids of the other species contained only 20% or less of these neutral lipids. Consistently, monogalactosyldiacylglycerols and phosphatidylethanol-amines were enriched in arachidonic acid and the galactolipids in eicosapentaenoic acid. The distribution of these acids in the phospholipids shows some preference for position 2. Together, the highly unsaturated C20 acids represented 80% of acyl groups in steryl esters. In triacylglycerols they were at average levels, while they were much less in sulfolipids and phosphatidylglycerols. Wax esters contained very little of the highly unsaturated acids but appreciable amounts of phytol and phytenic acid were found as wax constituents.     

Lipid structure and the behavior of cholesteryl esters in monolayer and bulk phases.

The behavior of cholesteryl esters at the air-buffer interface was studied as a function of molecular area and the presence of noncholesterol-containing lipids (colipids). The data obtained indicate that cholesteryl esters with other than long, saturated acyl groups can be present in surface phases up to packing densities approximately those in natural membranes. Their apparent molecular areas in such phases, which are largely determined by colipid structure, suggest their orientation with the ester function toward the interface. The extent of miscibility in the surface phase is also a strong function of colipid structure. Reversibility of the monolayer to bulk phase transition is determined exclusively by the acyl structure of the cholesteryl ester. Of the esters examined, only those with cis unsaturation collapsed reversibly. Our data predict that cholesteryl esters should be present in small, but finite amounts on the surface of arterial lipid deposits and that a prerequisite for the removal of such deposits is that the bulk lipid phase be in a liquid or liquid crystalline state.     

Fatty acids of astaxanthin esters in krill determined by mild mass spectrometry

Krill is a major source of astaxanthin, which has strong antioxidant activity. Fractions with astaxanthin monoesters and diesters of Antarctic krill Euphausia superba were isolated. Astaxanthin esters were separated by C18-HPLC depending on the number of carbons and double bonds of esterified fatty acid(s). Small amounts of other lipids remained in the samples, but relative molecular masses of carotenoid esters could be measured by field desorption mass spectrometry without fragmentation and interference from contaminant lipids. The fatty acids were determined by calculation of difference between astaxanthin and astaxanthin esters. Only five kinds of fatty acids, dodecanoate, tetradecanoate, hexadecanoate, hexadecenoate and octadecenoate, were detected. Fast atom bombardment mass spectrometry and secondary ion mass spectrometry showed similar spectra. The fatty acid composition in astaxanthin esters was different from those in krill lipids. Therefore, determination of fatty acids in carotenoid esters by a combination of HPLC elution profile and mild mass spectrometry is found to be a useful tool.     

Prenyl lipid formation in spinach chloroplasts and in a cell-free system of Synechococcus (Cyanobacteria): polyprenols,chlorophylls, and fatty acid prenyl esters

Isolated chloroplasts from spinach leaf cells, chloroplast subfractions, and a cell-free system of the cyanobacterium Synechococcus CCAP 6312 incorporated [1-¹⁴C]isopentenyl pyrophosphate in high yields into prenyl lipids. Products were polyprenols (C₂₀, C₄₅) chlorophylls, quinoid compounds, and fatty acid prenyl esters; prenyl pyrophosphates occurred in trace amounts, and carotenes were only formed to a limited extent in the Synechococcus system. The formation of fatty acid prenyl esters, which is described here for the first time, was found to occur in two different ways in the chloroplast system; by an acyl-CoA: polyprenol acyltransferase reaction associated with the envelope membranes and by a transesterification reaction from chlorophyll associated with the thylakoids. Endogenous fatty acid prenyl esters made up about 3% by weight of total lipids in spinach chloroplasts and were also found to be natural constituents of the cyanobacterial cells.Abbreviations Chl chlorophyll - Chl_GG chlorophyll a containing a geranylgeranyl side chain - IPP isopentenyl pyrophosphate     

Nitric oxide assisted hydrolysis of nitrophenylphosphate

In this study the use of sodium nitroprusside as nitric oxide donor in solutions was utilized. It has been established that maximum release of nitric oxide is achieved under irradiation by UV light at 254 nm. The synergistic effects of cobalt trimethylene diamine and nitroprusside towards the hydrolysis of nitrophenylphosphate under the above conditions for different cobalt to nitrophenylphosphate ratio were investigated. This study demonstrates that besides the effect of hydroxyl radicals, the direct interaction of nitric oxide with the phosphorous center also play a role in decontamination reactions of poorly biodegradable phosphate esters in natural waters, due to phototransformation.     

Surface skin lipids of birds — a proper host kairomone and feeding inducer in the poultry red mite,Dermanyssus gallinae

A factor in bird skin responsible forD. gallinae feeding on its natural hosts was investigated. Both skin and plumage of chickens contained substance(s) attractive to mites which was easily dissolved in benzene derivates, amyl acetate or ethyl acetate, thermostabile up to 100°C, nonvolatile, alkaline hydrolysable, and susceptible to oxidation. Components of surface skin lipids were ascertained to be the host-markers when using Chromatographic separation and consecutive in vitro feeding technique for testing the isolates. Purified specific fowl diol esters of fatty acids prepared from the secretion of uropygial (preen) glands of hens were at least as effective a feeding stimulant for the mites as the natural extract of surface lipids of birds.     

Production of new unsaturated lipids during wood decay by ligninolytic basidiomycetes

Lipids were analyzed by gas chromatography-mass spectrometry for a 7-week in vitro decay of eucalypt wood by four ligninolytic basidiomycetes. The sound wood contained up to 75 mg of lipophilic compounds per 100 g of wood. Hydrolysis of sterol esters, which represented 38% of total wood lipids, occurred during the fungal decay. The initial increase of linoleic and other free unsaturated fatty acids paralleled the decrease of sterol esters. Moreover, new lipid compounds were found at advanced stages of wood decay that were identified from their mass spectra as unsaturated dicarboxylic acids consisting of a long aliphatic chain attached to the C-3 position of itaconic acid. These dicarboxylic acids were especially abundant in the wood treated with Ceriporiopsis subvermispora (up to 24 mg per 100 g of wood) but also were produced by Phlebia radiata, Pleurotus pulmonarius, and Bjerkandera adusta. We hypothesize that three main alkylitaconic acids (tetradecylitaconic, cis-7-hexadecenylitaconic, and hexadecylitaconic acids) are synthesized by fungi in condensation reactions involving palmitic, oleic, and stearic acids. We suggest that both wood unsaturated fatty acids (present in free form or released from esters during natural decay) and unsaturated metabolites synthesized by fungi could serve as a source for peroxidizable lipids in lignin degradation by white rot basidiomycetes.     

Waxes and lipids associated with the external waxy structures of nymphs and pupae of the giant whitefly, Aleurodicus dugesii

The nymphs and pupae of the giant whitefly, Aleurodicus dugesii, produce large quantities of external lipids, both as waxy particles and as waxy filaments. The nymphs and pupae extrude filaments from two dorsal rows of five pores each. Filaments can attain lengths of 5-8 cm. The external lipids of nymphs and pupae consist largely of long-chain aldehydes, alcohols, acetate esters and wax esters. Hydrocarbons are minor components. Soon after hatching, the nymph produced an unidentified waxy fringe extruded laterally from its margin. After molting to the second instar, long, hollow, waxy filaments were produced by the immature stages. The major lipid class associated with the filaments was saturated wax esters (89%), mainly C44, C46 and C60. Associated with formation of the filaments were waxy particles in the shape of curls, which peeled off of the extruding filaments. Similar but more tubular-shaped curls were also produced by numerous lateral pores so that, eventually, the curls completely camouflaged the nymph. The major lipid class of the curls was wax esters (50%), mainly C44 and C46. The cuticular surface lipids of the nymphs were mainly long-chain aldehydes (43%) and wax esters (27%). Unsaturated fatty acid moieties constituted 2 and 19% of the wax esters of curls and nymph cuticular surface lipids, respectively. The major lipid classes of pupae and of their palisade were long-chain aldehydes and alcohols. No unsaturated wax esters were detected in the filaments, but 30% of pupal and 21% of palisade surface wax esters were unsaturated in their fatty acid moieties, 16:1, 18:1 and 20:1.     

Early alterations induced by carbon tetrachloride in the lipids of the membranes of the endoplasmic reticulum of the liver cell I. Separation and partial characterization of altered lipids

Alterations induced by carbon tetrachloride poisoning in fatty acids of liver microsomal lipids were studied. Thin layer chromatography of fatty acid methyl esters prepared from liver microsomal lipids, revealed, in the CCl₄-treated rats, the presence of a component (the “D” spot) with an R_f value lower than that of the methyl esters. The lipids recovered from this component showed a marked diene conjugation absorption when examined spectrophotometrically over the UV range, while the lipids recovered from the spot of the methyl esters showed no absorption of conjugated dienes.Studies carried out with labelled carbon tetrachloride indicated that compounds present in the “D” spot contained 28% of ¹⁴C applied to the chromatoplate. The spot of the methyl esters (the “M” spot) contained 42% of ¹⁴C applied to the chromatoplate. However, specific activity of the “D” spot was about 1000 times greater than specific activity of the “M” spot.The lipids recovered from either the “D” spot or the spot of the methyl esters were analyzed separately by gas-liquid chromatography (GLC) with an electron capture detector (ECD). It was found that the lipids recovered from the “D” spot showed no response, while those recovered from the spot of the methyl esters exhibited the response of the ECD, which was similar to that observed with the unfractionated fatty acid methyl esters. The lack of the response of the ECD for compounds in the “D” spot appears to be due to the fact that they cannot be eluted from the column.On the basis of the analytical results, it can be postulated that the “D” spot contains compounds formed by a chain termination addition reaction of free radicals derived from CCl₄ (probably trichloromethyl free radicals) to fatty acid free radicals containing conjugated dienes. On the other hand, the spot of the methyl esters appears to contain also, together with unmodified fatty acids, the fatty acids in which a simple addition of CCl₄ free radicals to double bonds has occurred.     

Comparative analysis of lipid composition in axenic strains of Blastocystis hominis.

1. Six axenic strains of Blastocystis hominis varied in content of lipids from 12 to 43 pg total lipid/cell. With all strains, phospholipid content was about 39% of total lipids. 2. Neutral lipid fractions of B. hominis were resolved into nine constituents, of which seven were identified tentatively. Sterol esters, principally esters of cholesterol, were the major neutral lipid constituent, accounting for 49-63% of the neutral lipids, and at least 30% of the total lipids. 3. Polar lipids were resolved into eleven constituents, of which nine were identified tentatively. Phosphatidylcholine was the major polar lipid constituent of all strains, accounting for 53-63% of the polar lipids, and about 22% of the total lipids.     

Retinyl esters form lipid droplets independently of triacylglycerol and seipin

Lipid droplets store neutral lipids, primarily triacylglycerol and steryl esters. Seipin plays a role in lipid droplet biogenesis and is thought to determine the site of lipid droplet biogenesis and the size of newly formed lipid droplets. Here we show a seipin-independent pathway of lipid droplet biogenesis. In silico and in vitro experiments reveal that retinyl esters have the intrinsic propensity to sequester and nucleate in lipid bilayers. Production of retinyl esters in mammalian and yeast cells that do not normally produce retinyl esters causes the formation of lipid droplets, even in a yeast strain that produces only retinyl esters and no other neutral lipids. Seipin does not determine the size or biogenesis site of lipid droplets composed of only retinyl esters or steryl esters. These findings indicate that the role of seipin in lipid droplet biogenesis depends on the type of neutral lipid stored in forming droplets.     

Oxidative degradation of model lipids representative for main paper pulp lipophilic extractives by the laccase–mediator system

Different model lipids-alkanes, fatty alcohols, fatty acids, resin acids, free sterols, sterol esters, and triglycerides-were treated with Pycnoporus cinnabarinus laccase in the presence of 1-hydroxybenzotriazole as mediator, and the products were analyzed by gas chromatography. The laccase alone decreased the concentration of some unsaturated lipids. However, the most extensive lipid modification was obtained with the laccase-mediator system. Unsaturated lipids were largely oxidized and the dominant products detected were epoxy and hydroxy fatty acids from fatty acids and free and esterified 7-ketosterols and steroid ketones from sterols and sterol esters. The former compounds suggested unsaturated lipid attack via the corresponding hydroperoxides. The enzymatic reaction on sterol esters largely depended on the nature of the fatty acyl moiety, i.e., oxidation of saturated fatty acid esters started at the sterol moiety, whereas the initial attack of unsaturated fatty acid esters was produced on the fatty acid double bonds. In contrast, saturated lipids were not modified, although some of them decreased when the laccase-mediator reactions were carried out in the presence of unsaturated lipids suggesting participation of lipid peroxidation radicals. These results are discussed in the context of enzymatic control of pitch to explain the removal of lipid mixtures during laccase-mediator treatment of different pulp types.     

Production of New Unsaturated Lipids during Wood Decay by Ligninolytic Basidiomycetes

Lipids were analyzed by gas chromatography-mass spectrometry for a 7-week in vitro decay of eucalypt wood by four ligninolytic basidiomycetes. The sound wood contained up to 75 mg of lipophilic compounds per 100 g of wood. Hydrolysis of sterol esters, which represented 38% of total wood lipids, occurred during the fungal decay. The initial increase of linoleic and other free unsaturated fatty acids paralleled the decrease of sterol esters. Moreover, new lipid compounds were found at advanced stages of wood decay that were identified from their mass spectra as unsaturated dicarboxylic acids consisting of a long aliphatic chain attached to the C-3 position of itaconic acid. These dicarboxylic acids were especially abundant in the wood treated with Ceriporiopsis subvermispora (up to 24 mg per 100 g of wood) but also were produced by Phlebia radiata, Pleurotus pulmonarius, and Bjerkandera adusta. We hypothesize that three main alkylitaconic acids (tetradecylitaconic, cis-7-hexadecenylitaconic, and hexadecylitaconic acids) are synthesized by fungi in condensation reactions involving palmitic, oleic, and stearic acids. We suggest that both wood unsaturated fatty acids (present in free form or released from esters during natural decay) and unsaturated metabolites synthesized by fungi could serve as a source for peroxidizable lipids in lignin degradation by white rot basidiomycetes.     

Characterization of Benthic Microbial Community Structure by High-Resolution Gas Chromatography of Fatty Acid Methyl Esters

Fatty acids are a widely studied group of lipids of sufficient taxonomic diversity to be useful in defining microbial community structure. The extraordinary resolution of glass capillary gas-liquid chromatography can be utilized to separate and tentatively identify large numbers of fatty acid methyl esters derived from the lipids of estuarine detritus and marine benthic microbiota without the bias of selective methods requiring culture or recovery of the microbes. The gas-liquid chromatographic analyses are both reproducible and highly sensitive, and the recovery of fatty acids is quantitative. The analyses can be automated, and the diagnostic technique of mass spectral fragmentation analysis can be readily applied. Splitless injection on glass capillary gas chromatographic columns detected by mass spectral selective ion monitoring provides an ultrasensitive and definitive monitoring system. Reciprocal mixtures of bacteria and fungi, when extracted and analyzed, showed progressive changes of distinctive fatty acid methyl esters derived from the lipids. By manipulating the environment of an estuarine detrital microbial community with antibiotics and culture conditions, it was possible to produce a community greatly enriched in eucaryotic fungi, as evidenced by scanning electron microscopic morphology. The fatty acid methyl esters from the lipids in the fungus-enriched detritus showed enrichment of the C₁₈ dienoic and the C₁₈ and C₂₀ polyenoic esters. Manipulation of the detrital microbiota that increased the procaryotic population resulted in an absence of large structures typical of fungal mycelia or diatoms, as evidenced by scanning electron microscopy, and a significantly larger proportion of anteiso- and isobranched C₁₅ fatty acid esters, C₁₇ cyclopropane fatty acid esters, and the cis-vaccenic isomer of the C₁₈ monoenoic fatty acid esters. As determined by these techniques, a marine settling community showed greater differences in bacterial as contrasted to microeucaryotic populations when compared with the microbial communities of benthic cores.     

Interaction of ceramides and tear lipocalin

The distribution of lipids in tears is critical to their function. Lipids in human tears may retard evaporation by forming a surface barrier at the air interface. Lipids complexed with the major lipid binding protein in tears, tear lipocalin, reside in the bulk (aqueous) and may have functions unrelated to the surface. Many new lipids species have been revealed through recent mass spectrometric studies. Their association with lipid binding proteins has not been studied. Squalene, (O-acyl) omega-hydroxy fatty acids (OAHFA) and ceramides are examples. Even well-known lipids such as wax and cholesteryl esters are only presumed to be unbound because extracts of protein fractions of tears were devoid of these lipids. Our purpose was to determine by direct binding assays if the aforementioned lipids can bind tear lipocalin. Lipids were screened for ability to displace DAUDA from tear lipocalin in a fluorescence displacement assay. Di- and tri-glycerides, squalene, OAHFA, wax and cholesterol esters did not displace DAUDA from tear lipocalin. However, ceramides displaced DAUDA. Apparent dissociation constants for ceramide-tear lipocalin complexes using fluorescent analogs were measured consistently in the submicromolar range with 3 methods, linear spectral summation, high speed centrifugal precipitation and standard fluorescence assays. At the relatively small concentrations in tears, all ceramides were complexed to tear lipocalin. The lack of binding of di- and tri-glycerides, squalene, OAHFA, as well as wax and cholesterol esters to tear lipocalin is consonant with residence of these lipids near the air interface.     

Effect of esters based on terpenoids and GABA on fluidity of phospholipid membranes

Abstract

The influence of esters based on gamma-aminobutyric acid (GABA) and mono-/bicyclic terpenoids on membrane structure was investigated. The mechanism of action for terpenoid esters on phospholipids of artificial membranes and lipids isolated from the rat stratum corneum was studied by fluorescence and FT-IR spectroscopy. We report here, that inclusion of monocyclic terpenoid esters in phospholipid liposomes leads to growth of excimer to monomer ratio (I_E/I_M) indicating a decrease of membrane microviscosity. Another mechanism of influence on biomembranes was proposed for ester of bicyclic borneol - in this case a high ratio of vibronic peak intensities (I₁/I₃) was revealed. The addition of terpenoid esters appears in the FT-IR spectra as intensity reduction of absorption bands associated with C?=?O, P?=?O and P–O–С groups of lecithin phospholipids. Similar results were obtained after esters addition to lipids isolated from stratum corneum indicating a decrease of hydrogen bonds number between polar groups of lipids. Thus, the influence of terpenoid esters on molecular organization of the lipid matrix substantiates the feasibility of their use after transdermal delivery in vivo.