Chemical Characterization of Lipid A from Some Marine Proteobacteria

Lipids A from type and wild strains of marine Proteobacteria belonging to Alteromonadaceae (Alteromonas (1 species), Idiomarina (1 species), and Pseudoalteromonas (8 species) genera) and Vibrionaceae (Shewanella (1 species) and Vibrio (1 species) genera) families and Marinomonas genus (1 species) were isolated by hydrolysis of their respective lipopolysaccharides with 1% acetic acid. Based on thin-layer chromatography data, the lipids A studied had low heterogeneity and generated family-specific patterns varying in numbers of bands and their chromatographic mobility. Total chemical analysis of the compounds showed that they contained glucosamine, phosphate, and fatty acids with decanoate (I. zobellii KMM 231^T lipid A) or dodecanoate (lipids A of the other bacteria) and 3-hydroxy alkanoates as the major fatty acid components. Unlike terrestrial bacterial lipids A, lipids A of marine Proteobacteria had basically monophosphoryl (except V. fluvialis AQ 0002B lipid A with its two phosphate groups) and pentaacyl (except S. alga 48055 and V. fluvialis AQ 0002B lipids A which were found to have six residues of fatty acids per molecule of glucosamine disaccharide) structural types, low toxicity, and may be useful as potential endotoxin antagonists.     

Characterization of lipid A and polysaccharide moieties of the lipopolysaccharides from Vibrio cholerae.

Lipid A and polysaccharide moieties obtained by mild acid hydrolysis of the lipopolysaccharides from Vibrio cholerae 569 B (Inaba) and Vibrio el-tor (Inaba) were characterized. Heterogeneity of lipid A fractions was indicated by t.l.c. and by gel filtration of the de-O-acylated products from mild alkaline methanolysis of the lipids. Presumably lipid A contains a glucosamine backbone, and the fatty acids are probably bound to the hydroxyl and amino groups of glucosamine residues. Approximately equal amounts of fatty acids C16:0, C18:1 and 3-hydroxylauric acid were involved in ester linkages, but 3-hydroxymyristic acid was the only amide-linked fatty acid. Sephadex chromatography of the polysaccharide moiety showed the presence of a high-molecular-weight heptose-free fraction and a low-molecular-weight heptose-containing fraction. Haemagglutination-inhibition assays of these fractions showed the heptose-free fraction to be an O-specific side-chain polysaccharide, whereas the heptose-containing fraction was the core polysaccharide region of the lipopolysaccharides. Identical results were obtained for both organisms.     

Synthesis of lipids from acetate by human preputial and abdominal skin in vitro

Lipogenesis in vitro from acetate-1-(14)C was studied in human preputial skin and abdominal skin. Radioactive lipids were separated by column chromatography on Florisil and by thin-layer chromatography on silica gel. Radioactivity was incorporated chiefly into the triglyceride, sterol, and polar lipid fractions, while lesser amounts of (14)C were found in the hydrocarbon, wax, diglyceride, monoglyceride, and fatty acid fractions; labeling of steryl esters was minimal. On thin-layer chromatography, the radioactive polar lipids had mobilities similar to lysolecithin, phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidic acid. The radioactive fatty acids of the different lipid fractions were separated by gas-liquid chromatography. The major (14)C-labeled acids were 16:0 and 18:0. Radioactivity was also detected in acids 14:0, 15:0, 16:1, 18:1, 18:2, 20:0, 20:1, 22:0, 24:0, 24:1, and 26:0. No radioactivity could be detected in arachidonic acid, although this fatty acid comprises 9% of the chromatographed fatty acids. The pattern of incorporated (14)C was different from the percentage mass composition of the fatty acids. Skin is therefore active in the biosynthesis of a wider variety of lipids than previously demonstrated.     

Detailed Analyses of Individual Neutral Lipid Classes of Pneumocystis

SUMMARY Individual neutral lipid classes of Pneumocystis carinii carinii were isolated and purifed by thin-layer chromatography. The fatty acids in steryl esters, triglycerides, free fatty acids, diglycerides, and monoglycerides were quantified by gas-liquid chromatography. The fatty acid compositions of these lipids were similar to those of the neutral lipid classes in whole rat lung controls, unlike comparable studies of phospholipid classes. The free fatty alcohols of rat lung controls and P. carinii were also identified and quantified; only saturated fatty alcohols were detected.     

Separation of neutral lipids and free fatty acids by high-performance liquid chromatography using low wavelength ultraviolet detection

Normal phase, isocratic high-performance liquid chromatography methods are described for the separation of neutral lipid and fatty acid classes using low wavelength detection. Prior to high-performance liquid chromatography, methods were developed and are described for the separation of phospholipids from neutral lipids and fatty acids using small (600 mg) silica Sep-PaksTM. Recoveries of cholesteryl esters, triglycerides, fatty acids, and phospholipids from the silica columns were greater than 95%. Two mobile phases are described for lipid class separation by high-performance liquid chromatography. The first mobile phase, hexane-2-propanol-acetic acid 100:0.5:01, resulted in incomplete separation of cholesteryl ester and triglyceride but excellent separations of fatty acids and cholesterol. The second mobile phase, hexane-n-butyl chloride-acetonitrile-acetic acid 90:10:1.5:0.01, resulted in complete separation of the four lipid classes. This mobile phase also separated individual triglycerides and fatty acids based on the number of double bonds. Recoveries of radiolabeled lipids for the four lipid classes from high-performance liquid chromatography was greater than 95% with both mobile phases.     

Characterization of the Lipids of Butyrivibrio fibrisolvens

Butyrivibrio fibrisolvens strain D-1 was grown on a lipid-free chemically defined medium. The lipids were extracted with chloroform-methanol and separated into nonpolar and polar fractions by silicic acid column chromatography. Further separations were made by preparative thin-layer chromatography. The lipid fractions were identified by specific staining reactions and R(F) values, by phosphorus and nitrogen determinations, by chromatography of hydrolysis products, and by the use of infrared spectroscopy. The major nonpolar lipid was free fatty acid. Four major polar lipids were identified: phosphatidylethanolamine, phosphatidyl glycerol, lipoaminoacid, and glycolipid. The lipoaminoacid contained alanine, leucine, and isoleucine. The glycolipid contained galactose. The major fatty acids identified were C16:0 and C18:1. The significance of the presence of lipoaminoacid is discussed.     

Isolation and composition of the carotenoid-containing oil droplets from cone photoreceptors.

A procedure for isolating the carotenoid-containing oil droplets of cone retinal photoreceptors of Gallus domesticus is described. The oil droplets, composed almost entirely of neutral lipids and carotenoids, have been separated into ten chromatographic components. Similar separations have been carried out on the total retinal neutral lipids for comparison. The neutral lipids represented 26.1% of the total retinal lipid. Cholesterol, cholesterol ester, mono-, di- and triacylglycerols represented 92.6% of the total neutral lipid. Each of these and other minor neutral lipid components were also present in the lipids extracted from the isolated oil droplets in correspondingly similar concentrations. However, the concentrations of carotenoids were greatly enriched in the neutral lipids of the oil droplets. Each of the major fatty acyl-containing neutral lipids from the chromatography of oil droplet lipids is greatly enriched in polyunsaturated fatty acids when compared with the corresponding component from the total neutral lipid chromatography. In the acylglycerols and free fatty acid fraction from the oil droplets, linoleic and arachidonic acid together represented 52-83% of the total polyunsaturated fatty acids present. The remainder was generally distributed about equally among six other acids. Except for the diacylglycerol fraction, linoleic acid was usually the most enriched acid in a specific oil droplet fraction when compared with any other polyunsaturated fatty acids. A similar pattern of polyunsaturated fatty acid enrichment observed in the fatty acids of the outer segment phospholipids relative to the corresponding total phospholipid fractions of this cone rich retina (Johnston, D. and Hudson, R.A. (1974) Biochim. Biophys. Acta 369, 269) suggest possible metabolic relationships between the oil droplet neutral lipids and the outer segment membrane phospholipids of the cone photoreceptors. A mechanism for the accumulation of the carotenoids in the oil droplets is also discussed.     

Changes in the Amount of Complex Lipids in the Seeds and in the Pericarp during the Development of Ivy Fruit (Hedera helix)

By a combination of thin-layer chromatography and gas liquid chromatography, a complete study of the development of the different lipid classes and of their fatty acids, during the development of the fruit of Hedera helix L., the English Ivy, has been achieved. In any part of the fruit observed, at any particular stage, the phospholipids and the neutral lipids are the most abundant lipid classes. They accumulate during the entire process of maturation, whereas significant changes occur in their relative proportions, phospholipids being largely dominant until fruit blackening. The accumulation of fatty acids during maturation is characterized by large amounts of C_18:1 in the neutral lipids, especially in the seed, where petroselinic acid (C_18:1Δ⁶) reaches 86% of the total fatty acids. To a smaller extent, the phospholipids also accumulate and thus have the character of reserve molecules. However, their composition remains more stable, which relates them to the “structural lipids” such as galactolipids that maintain their characteristic fatty acid composition, despite the radical changes occurring in the fatty acid metabolism during fruit ripening.     

Analysis of lipids by gas-liquid chromatography and complementary methods in four strains of Aedes aegypti mosquitoes

Fatty acid composition of total lipids, neutral lipids and phospholipids of strains of Aedes aegypti were determined. The fatty acid composition of the strains differed quantitatively with regard to the relative percentage of commonly occurring fatty acids. Gas-liquid chromatography of fatty acid methyl esters showed 18:1 (oleic or elaidic) to be the predominant fatty acid. The fatty acid was identified as oleic by argentation thin-layer chromatography. A modified colorimetric method was used to determine tissue-free fatty acids. The lipids were predominantly triacylglycerol with lesser amounts of free fatty acids and decreasing amount of sterol ester, sterol, monoacylglycerol, diacylglycerol and hydrocarbons. The data show considerable lipid differences between the Caribbean strains (Les Cayes, Haiti, and San Juan, Puerto Rico) and the Jakarta (Indonesia) strain. The Shimba Hills (Kenya) strain was more similar to Jakarta than to the Caribbean strains. The results obtained with the different strains are discussed in relation to the established oral susceptibility to Dengue 1 and Dengue 2, yellow fever, and genetic analysis by isoenzyme studies.     

Evidence for a Composite State of an F′his,gnd Element and a Cryptic Plasmid in a Derivative of Salmonella typhimurium LT2

The zoospores of Blastocladiella emersonii, when derived from cultures grown on solid media, contain about 11% total lipid. This lipid was separated chromatographically on silicic acid into neutral lipid (46.6%), glycolipid (15.8%), and phospholipid (37.6%). Each class was fractionated further on columns of silicic acid, Florisil, or diethylaminoethyl-cellulose, and monitored by thin-layer chromatography. Triglycerides were the major neutral lipids, mono- and diglycosyldiglycerides were the major glycolipids, and phosphatidylcholine and phosphatidylethanolamine were the major phospholipids. Other neutral lipids and phospholipids detected were: hydrocarbons, free fatty acids, free sterols, sterol esters, diglycerides, monoglycerides, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidic acid, phosphatidylserine, and phosphatidylinositol. Palmitic, palmitoleic, stearic, oleic, gamma-linolenic, and arachidonic acids were the most frequently occurring fatty acids. When B. emersonii was grown in (14)C-labeled liquid media, lipid again accounted for 11% of both mature plants and zoospores released from them. The composition of the lipid extracted from such plants and spores was also the same; however, it differed markedly from that of the lipid in spores harvested from solid media, consisting of 28.3% neutral lipid, 12.0% glycolipid, and 59.7% phospholipid. The major lipids were again triglycerides for neutral lipids, mono- and diglycosyldiglycerides for glycolipids, and phosphatidyl choline and phosphatidylethanolamine for phospholipids.     

Seasonal variation in lipid and fatty acid composition of ice algae from the Barents Sea

The fatty acid compositions of neutral lipid, glycolipid and phospholipid fractions from ice algae sampled from the Barents Sea in spring and autumn were examined for seasonal differences. The ice-algal assemblages were dominated by diatoms. In spring, Nitzschia frigida was the most common species whereas resting stages of Thalassiosira bioculata and Actinocyclus cf curvatulus predominated in autumn. With the exception of one spring sample, neutral lipids predominated over glycolipids and phospholipids in all algal samples. The lipid fractions displayed characteristic fatty acid compositions. In the spring samples the major fatty acids of the neutral lipid fraction were 16:0, 16:1(n-7) and 20:5(n-3) whilst the glycolipid fraction was characterised by higher levels of 20:5(n-3) and C16 polyunsaturated fatty acids, particularly 16:4(n-1). Phospholipids contained higher levels of 22:6(n-3) than the other two lipid fractions although 20:5(n-3) was still the major polyunsaturated fatty acid. In the autumn samples, the neutral lipid fraction contained higher proportions of saturated fatty acids and 16:1(n-7) than the two polar lipid fractions and 22:6(n-3) was most abundant in phospholipids. As with the spring samples, 20:5(n-3) was the major polyunsaturated fatty acid in all lipid fractions of the autumn algae. Overall, the fatty acid compositions of the lipid fractions from spring and autumn algal samples were similar and are consistent with diatoms being the predominant group in the ice algae studied. The high level of neutral lipids observed in both spring and autumn samples suggests that the production of neutral lipids is characteristic of ice algae regardless of season. Nevertheless, some species-specific differences in lipid production may exist since the neutral lipid content of autumn samples containing mainly A. curvatulus was substantially higher than those in which T. bioculata predominated. Received: 26 September 1997 / Accepted: 12 January 1998     

The effects of branched chain fatty acid incorporation into Neurospora crassa membranes

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), an unusual branched chain fatty acid thought to disrupt the hydrophobic regions of membranes, can be incorporated into the lipids of growing Neurospora cultures. The phytanic acid must be supplied in a water soluble form, esterified to a Tween detergent (Tween-Phytanic). This fatty acid and its oxidation product, pristanic acid, were found in both the phospholipid and neutral lipid fractions of Neurospora. In phospholipids of the wild-type strain, phytanic acid was present to the extent of 4 to 5 moles percent of the fatty acids and pristanic acid, about 41 moles percent. The neutral lipids contained 42 and 4 moles percent of phytanic and pristanic acids respectively. By employing a fatty acid-requiring mutant strain (cel^?), the phytanic acid level was raised to a maximum of 16 moles percent in the phospholipids and to 63 moles percent in the neutral lipids. Under this condition, the level of pristanic acid was reduced to about 6 moles percent in phospholipids and 1 mole percent in the neutral lipids. The phytanic acid levels could not be further elevated by increased supplementation with phytanic acid or by a change in the growth temperature. In strains with a high phytanic acid content, the complete fatty acid distribution of the phospholipids and neutral lipids was determined. In the neutral lipids, phytanic acid appeared to replace the 18 carbon fatty acids, particularly linoleic acid. The presence of phytanic acid in the phospholipids was confirmed by mass spectrometry, and by the isolation of a phospholipid fraction containing this fatty acid via silicic acid column chromatography. Most of the phytanic acid in phospholipids appeared to be in phosphatidylethanolamine, and 2 lines of evidence suggest that it was esterified to both positions of this molecule. In the fatty acid-requiring mutant strain (cel^?), the replacement by phytanic acid of 10 to 15% of the fatty acids in the phospholipid produced an aberrant morphological change in the growth pattern of Neurospora and caused this organism to be osmotically more fragile than the wild-type strain. The lack of noticeable effect of the high levels of pristanic acid in the phospholipids suggests that it is not just the presence of the methyl groups in a branched chain fatty acid which leads to the altered membrane function in this organism.     

Lipolysis by Intracellular Lipase of Streptococcus lactis against Its Neutral Lipids Obtained by Growth at Low Temperature

Lipolytic activities of intracellular lipase obtained from Streptococcus lactis 527 cells grown at 30°C were determined using bacterial neutral lipids extracted from cells grown at 10 and 30°C. The amounts of free fatty acids liberated from lipids by lipase were in the order: 30°C neutral lipid > 10°C neutral lipid > triolein > intracellular membrane fraction. Glycerides hydrolyzed partially by lipase were detected on thin-layer plates and were composed of 1,3- and 1,2-diglycerides, fatty acids and unhydrolyzed triglycerides. Fatty acids liberated from neutral lipids by lipase were determined by gas chromatography. It was found that the major acid was cy-C₁₀ and the minor among the acids liberated from 10°C neutral lipid, whereas the major acid was and the minors and cy-C₁₀ from 30°C lipid.     

Critical Assessment of Glyco- and Phospholipid Separation by Using Silica Chromatography

Phospholipid-derived fatty acids (PLFAs) are commonly used to characterize microbial communities in situ and the phylogenetic positions of newly isolated microorganisms. PLFAs are obtained through separation of phospholipids from glycolipids and neutral lipids using silica column chromatography. We evaluated the performance of this separation method for the first time using direct detection of intact polar lipids (IPLs) with high-performance liquid chromatography–mass spectrometry (HPLC-MS). We show that under either standard or modified conditions, the phospholipid fraction contains not only phospholipids but also other lipid classes such as glycolipids, betaine lipids, and sulfoquinovosyldiacylglycerols. Thus, commonly reported PLFA compositions likely are not derived purely from phospholipids and perhaps may not be representative of fatty acids present in living microbes.     

Thehalose mycolates from Nocardia asteroides,Nocardia farcinica,Gordona lentifragmenta and Gordona bronchialis

Isolation of glycolipids from Nocardia asteroides, N. farcinica, Gordona lentifragmenta and G. bronchialis, by column chromatography of lipid extracts on a 50% (w/w) mixture of silicic acid and silica gel H, is described. The isolated materials were partially characterized by infrared spectroscopy, optical rotation and refractive index measurements, and by identifying the products of alkaline hydrolysis. Analytical studies showed that the glycolipids released only trehalose in the aqueous phase while mycolic acids were the constituent fatty acids identified.The isolated lipids are trehalose esters in which the trehalose molecule is esterified with mycolic acids.     

Fatty acids synthesized from hexadecane by Pseudomonas aeruginosa

Romero, Ethel M. (Universidad Nacional de la Plata, La Plata, Argentina), and Rodolfo M. Brenner. Fatty acids synthesized from hexadecane by Pseudomonas aeruginosa. J. Bacteriol. 91:183-188. 1966.-The lipids extracted from Pseudomonas aeruginosa incubated with hexadecane in a mineral medium were separated into a nonpolar and three polar fractions by thin-layer chromatography. The fatty acid composition of the four cellular fractions and that of the lipids excreted into the medium was studied by gas-liquid chromatography. Saturated fatty acids with 14 to 22 carbons were recognized, together with monoenoic, dienoic, and hydroxylated acids. Hydroxylated fatty acids were principally found in two polar fractions containing rhamnose and glucose; the other polar fraction, containing serine, alanine, ethanolamine, and leucine, was richer in monoenoic fatty acids. Octadecadienoic acid was found in the neutral fraction.     

Induction of tryptophan oxygenase and tyrosine aminotransferase by metabolites of hydrocortisone

Metabolites of hydrocortisone were isolated from rat liver on a preparative scale, fractionated by column chromatography on Sephadex LH-20 and silica gel and tested for biological activity. Apart from the well known neutral metabolites, steroid glucuronides and sulfates, we obtained metabolite fractions containing non-conjugated steroidal carboxy acids and acid metabolites of unknown structure. One of these fractions induced tyrosine aminotransferase (EC 2.6.1.5) in adrenalectomized female rats but not trptophan oxygenase (EC 1.13.11.11), whereas another one mainly increased activity of tryptophan oxygenase. The doses necessary to significantly induce both enzymes were much lower in case of these metabolites than in the case of hydrocortisone itself. The active fractions eluting from silica gel column were analyzed by thin-layer chromatography in two different solvent systems. Absence of hydrocortisone in these fractions could be clearly demonstrated. Furthermore, the active fractions eluting from the silica gel column were characterized by treatment with an extract from Helix pomatia and/or diazomethane and subsequent analysis by thin-layer chromatography. We conclude, considering the biological activity of some synthesized derivatives of hydrocortisone, that the biologically active components are acid metabolites of hydrocortison which are not identical to any of the known metabolites.     

Isolation and Characterization of a Neutral Glycosphingolipid from the Epimastigote Form of Trypanosoma mega1

ABSTRACT. A glycosphingolipid fraction from Trypanosoma mega was isolated after acetylation and was further purified on a silicic acid column. Final purification was by preparative thin-layer chromatography. The carbohydrate components of the glycolipid were fucose and galactose in approximately equimolar amounts. The neutral glycolipid of T. mega has a sphingosine base composition that consists of sphingosine and traces of dihydrosphingosine. Fatty acids forming amide groups with the sphingosine bases were analyzed by gas-liquid chromatography-mass spectrometry and are a mixture of normal and α-hydroxy fatty acids. Normal C_16:0, C_18:0, and 2-hydroxy C_18:0 are the predominant fatty acids.     

Biochemical studies on the cell wall lipopolysaccharides (O-antigens) of Vibrio cholerae 569 B (Inaba) and El-tor (Inaba).

Lipopolysaccharides were isolated from the cell walls of Vibrio cholerae 569 B (Inaba) and El-tor (Inaba). Chemical analysis revealed the presence of glucose, fructose, mannose, heptose, rhamnose, ethanolamine, fatty acids and glucosamine. The lipopolysaccharides do not contain 2-keto-3-deoxyoctonate, the typical linking sugar of polysaccharide and lipid moieties of enterobacterial lipopolysaccharides. Galactose, a typical core polysaccharide component of many gram-negative bacteria was also absent from lipopolysaccharides of these organisms. By hydrolysis in 1% acetic acid, the lipopolysaccharides have been separated into a polysaccharide part (degraded polysaccharide) and a lipid part (lipid A). Components of degraded polysaccharide and lipid A moiety were identified and determined. The lipid A fractions contained fatty acids, phosphorus and glucosamine. All the neutral sugars detected in lipopolysaccharides were shown to be the constituents of its polysaccharide moiety. The fatty acid analysis of lipopolysaccharide and lipid A showed the presence of both hydroxy and non hydroxy acids. They were different from those of lipids extracted from cell walls before the extraction of lipopolysaccharides. 3-Hydroxylauric and 3-hydroxymyristic acids predominated in lipopolysaccharide and lipid A of Vibrio cholerae and El-tor (Inaba).     

Carbohydrate and lipid components of hyphae and conidia of human pathogenFonsecaea pedrosoi

The carbohydrate and lipid components of mycelium and conidia ofFonsecaea pedrosoi (Brumpt) were analysed by paper, thin-layer and gas-chromatography, mass spectrometry and ultraviolet spectroscopy. Glucose, mannose, galactofuranose, rhamnose and glucosamine were polysaccharide components identified inF. pedrosoi. Significant changes in the carbohydrate pattern occurred during the conversion of mycelium into conidia. Rhamnose was predominant in conidia whereas galactose was prominent in mycelium. Palmitic, stearic, oleic, linoleic, and arachidonic acids were the fatty acids identified in the total lipid fraction. Palmitic and oleic acids were major fatty acids. Marked alterations in the fatty acid constituents were observed between the cell types ofF. pedrosoi. Arachidonic acid was detected only in conidia and linoleic acid was preferentially identified in mycelium. Differences in the sterol composition was also associated with morphogenesis inF. pedrosoi. Two main sterols, ergosterol and another less polar sterol, not fully characterized, were found in mycelium whereas in conidia only the latter sterol was present.