Optical Configuration Analysis of Hydroxy Fatty Acids in Bacterial Lipids by Chiral Column High-Performance Liquid Chromatography

Through the adoption of a chiral stationary phase in high-performance liquid chromatography and a simple derivatization method for hydroxy fatty acids, it became easy to separate and identify the optical isomers of 2- and 3-hydroxy fatty acids composing several kinds of microbial lipids. The 2- and 3-hydroxy fatty acids were converted with dinitrophenyl isocyanate to their 3, 5-dinitrophenyl urethane derivatives (DU-derivatives), which were analyzable by HPLC using a chiral column. By varying the composition of an eluent, separation of the DU-derivatives of hydroxy fatty acids differing in optical configuration, chain length and position of hydroxyl group was achieved. The general elution orders of these DU-derivatives were determined with authentic 2- and 3-hydroxy fatty acids. Small amounts (~300 μg) of ornithine-containing lipids isolated from the Serratia marcescens strains were examined by this method to identify 3-hydroxy fatty acids of the lipids as D isomers.     

Biophysical properties of tear film lipid layer II. Polymorphism of FAHFA

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered class of endogenous lipids that consist of two acyl chains connected through a single ester bond. Being a unique species of FAHFAs, (O-acyl)-ω-hydroxy fatty acids (OAHFAs) differ from other FAHFAs in that their hydroxy fatty acid backbones are ultralong and their hydroxy esterification is believed to be solely at the terminal (ω-) position. Only in recent years with technological advances in lipidomics have OAHFAs been identified as an important component of the tear film lipid layer (TFLL). It was found that OAHFAs account for approximately 4 mol% of the total lipids and 20 mol% of the polar lipids in the TFLL. However, their biophysical function and contribution to the TFLL is still poorly understood. Here we studied the molecular biophysical mechanisms of OAHFAs using palmitic-acid-9-hydroxy-stearic-acid (PAHSA) as a model. PAHSA and OAHFAs share key structural similarities that could result in comparable biophysical properties and molecular mechanisms. With combined biophysical experiments, atomic force microscopy observations, and all-atom molecular dynamics simulations, we found that the biophysical properties of a dynamic PAHSA monolayer under physiologically relevant conditions depend on a balance between kinetics and thermal relaxation. PAHSA molecules at the air-water surface demonstrate unique polymorphic behaviors, which can be explained by configurational transitions of the molecules under various lateral pressures. These findings could have novel implications in understanding biophysical functions that FAHFAs, in general, or OAHFAs, specifically, play in the TFLL.     

Existence of carotenoids in Acholeplasma axanthum.

The neutral lipids of Acholeplasma axanthum contain carotenoid pigments, as evidenced by spectral characteristics, visual color, color reactions, and labeling with [2-14C-A1mevalonic acid. Approximately 80% of the label from [2-14C]mevalonic acid appeared in esterified fatty acids of the glycolipids and polar lipids. These carboxylic acids behaved as hydroxy acids of varying chain length.     

Antioxidant effect of anthocyanin on enzymatic and non-enzymatic lipid peroxidation.

In vitro enzymatic and non-enzymatic polyunsaturated fatty acid peroxidation was significantly inhibited in a dose dependent manner by purified anthocyanin, a deep-red colour pigment from carrot cell culture. The kinetics showed that anthocyanin is a non-competitive inhibitor of lipid peroxidation. Anthocyanin has been found to be a potent antioxidant compared to classical antioxidants such as butylated hydroxy anisole (BHA), butylated hydroxy toulene (BHT) and alpha tocopherol. This natural agent, in addition to imparting colour to the food, might prevent autooxidation of lipids as well as lipid peroxidation in biological systems.     

Dietary hydroxy fatty acids are absorbed in humans: implications for the measurement of 'oxidative stress' in vivo.

Lipid peroxidation products formed in vivo or originating from the diet may lead to atherosclerosis. However, little is known about the absorption of these products in man. We studied the absorption of fat (30 g) containing 14-15 mg [U-13C]-labeled hydroxy or dihydroxy triglycerides in two groups of six apparently healthy women aged 40 +/- 2 years. Post-prandial 13C-labeled hydroxy fatty acid concentration increased in a pattern somewhat different from that of plasma triglycerides, with peak levels being reached between 4 and 6 h. However, the amount of 13C-labeled oxidized fat absorbed (area under the curve of plasma concentrations from 0 to 8 h) was related to that of plasma triglycerides: 13C hydroxy vs TG (r = 0.88, p <.02), and 13C dihydroxy vs TG (r = 0.85, p <.05). 13C monohydroxy triglycerides appeared to be absorbed to a greater extent than those of 13C dihydroxy triglycerides. Although low levels of 13C hydroxy lipids could be detected in fasting plasma after 24 h, concentrations were very low. Dietary lipid oxidation products are absorbed. The measurement of hydroxy fatty acids in plasma total lipids may not be a valid marker of lipid peroxidation in vivo when subjects are not fasting.     

An improved flame ionization detector for high performance liquid chromatography

A flame ionization detector and associated transport device of high sensitivity for liquid chromatography are described. The system is based on the moving chain principle and employs a stainless steel belt of perforated structure that confers on it superior surface properties for collection and transport of column eluent. After evaporation of the solvent, the solute is converted to hydrocarbons in a stainless steel reactor of sandwich construction and is combusted in a hydrogen flame. The universality of the system is demonstrated with albumin and glucose, as well as with lipids. Sensitivity and reproducibility of the system are demonstrated also with a standard mixture of neutral lipids.     

Abundance of lipids in differently sized aggregates depends on their chemical composition

Evidence for a vital role of soil mineral matrix interactions in lipid preservation is steadily increasing. However, it remains unclear whether solvent-extractable (‘free’) or hydrolyzable (‘bound’) lipids, including molecular proxies, e.g., for cutin and suberin, are similarly affected by different stabilization mechanisms in soil (i.e., aggregation or organo-mineral association). To provide insights into the effect of these stabilization mechanisms on lipid composition and preservation, we investigated free and bound lipids in particulate and mineral soil fractions, deriving from sand- and silt-/clay-sized aggregates from a forest subsoil. While free lipids accumulated in sand-sized aggregates, the more complex bound lipids accumulated in silt- and clay-sized aggregates, particularly in the respective mineral fractions?<?6.3 µm (fine silt and clay). The presence of both, cutin and suberin markers indicated input of leaf- and root-derived organic matter to the subsoil. Yet, our cutin marker (9,10,ω-trihydroxyoctadecanoic acid) was not extracted from the mineral aggregate compartments?<?6.3 µm, perhaps due to its chemical structure (i.e., cross-linking via several hydroxy groups, and thus higher ‘stability’, in macromolecular structures). Combined, these results suggest that the chemical composition of lipids (and likely also that of other soil organic matter compounds) governs interaction with their environment, such as accumulation in aggregates or association with mineral soil compartments, and thus indirectly influences their persistence in soil.     

The mechanism of oleic acid nitration by *NO(2)

Fatty acid nitration is a recently discovered process that generates biologically active nitro lipids; however, its mechanism has not been fully characterized. For example, some structural details such as vinyl and allyl isomers of the nitro fatty acids have not been established. To characterize lipids that originated from a biomimetic reaction of *NO(2) with oleic acid, we synthesized several isomers of nitro oleic acids and studied their chromatography and mass spectra by various techniques of mass spectrometry. LC/MS analysis performed on a high resolution micro column detected molecular carboxylic anions of various oleic acid nitro isomers (NO(2)OA). Esterification of NO(2)OA with pentafluorobenzyl bromide and diisopropylethylamine as a catalyst produced a unique isoxazole ester derivative exclusively from allyl NO(2)OA isomers via dehydration of the nitro group at ambient temperatures. This new analytical procedure revealed that *NO(2) generated two vinyl and two allyl isomers of NO(2)OA. The vinyl isomers showed high regioselectivity with the 1.8:1 preference for the 10-NO(2)OA isomer that was absent among allylic isomers. The nitration also generated elaidic acid via cis-trans isomerization and diatereoisomers of vicinal nitro hydroxy, nitro keto and alpha-nitro epoxy stearic acids with high stereo and regioselectivity. Nitration of small unilamelar phospholipid vesicles resulted in several phospholipids containing nitro lipids and elaidic acid amenable to hydrolysis by phospholipase A(2).     

Influence of exogenous natural oils on the omega-1 and omega-2 hydroxy fatty acid moiety of sophorose lipid produced by Candida bombicola

Candida bombicola can synthesize monohydroxy fatty acid as a moiety of sophorose lipids. The hydroxy fatty acids contained in a major lactone were identified by GC-MS, after culturing with natural oils such as coconut, rapeseed, olive, and soybean oils. Hydroxy fatty acids of C18 and C16 were always synthesized, but differences were observed among the oils regarding the positions of hydroxyl groups, unsaturation, and composition of the fatty acids. A new C17 hydroxy acid was found without addition of oil.     

Phospholipid analogues of Porphyromonas gingivalis

Porphyromonas has lipids containing hydroxy acids and C16:0 and iso-C15:0 major monocarboxylic acids among others. Nothing is known of its individual phospholipid molecular species. The aim of this study was to determine molecular weights and putative identities of individual phospholipid molecular species extracted from Porphyromonas gingivalis (seven strains), P. asaccharolytica (one strain) and P. endodontalis (two strains). Cultures on Blood-Fastidious Anaerobe Agar were harvested, washed and freeze-dried. Phospholipids were extracted and separated by fast atom bombardment mass spectrometry (FAB MS) in negative-ion mode. Phospholipid classes were also separated by thin layer chromatography (TLC). The major anions in the range m/z 209-299 were consistent with the presence of the C13: 0, C15: 0, C16: 0 and C18: 3 mono-carboxylate anions. Major polar lipid anion peaks in the range m/z 618-961 were consistent with the presence of molecular species of phosphatidylethanolamine, phosphatidylglycerol and with unidentified lipid analogues. Porphyromonas gingivalis differed from comparison strains of other species by having major anions with m/z 932, 946 and 960. Unusually, a feline strain of P. gingivalis had a major peak of m/z 736. Selected anions were studied by tandem FAB MS which revealed that peaks with m/z 653 and 946 did not correspond to commonly occurring classes of polar lipids. They were however, glycerophosphates. It is concluded that the polar lipid analogue profiles obtained with Porphyromonas are quite different from those of the genera Prevotella and Bacteroides but reveal heterogeneity within P. gingivalis.     

Occurrence of Phosphonosphingolipids in Bdellovibrio bacteriovorus Strain UKi2

The major phospholipids of two strains of Bdellovibrio bacteriovorus were characterized. Both strain UKi1, which is obligately saprophytic, and strain UKi2, which is facultatively parasitic, contained phosphatidylethanolamine and phosphatidylglycerol as their major glycerophosphatides. A branched, 15-carbon fatty acid is the major component of these alkali-labile lipids. Absent from UKi1 but present in UKi2 were three alkali-stable lipids (compounds 8, 9, and 11) which appear to be phosphosphingolipids. After acid hydrolysis, both compound 8 and 9 yield the identical phosphorus-containing substance that is water soluble, dipolar ionic, and ninhydrin positive. This substance appears to contain a C-P bond since P(i) could not be released from this substance by treatment with alkaline phosphatase or by very harsh mineral acid treatment. Based on chromatographic comparisons, this phosphonate appears to be a novel lipid constituent. Upon degradation, compound 8 yields 1 mol of dihydroxy long-chain base and compound 9 yields 1 mol of a trihydroxy long-chain base. These bases appear to have a 17-carbon, possibly branched, structure based on gas-liquid chromatography retention times. Degradation of both sphingolipids yields a mixture of hydroxy fatty acids, the major component being a branched, 15-carbon hydroxy acid.     

Influence of Exogenous Natural Oils on the ω-1 and ω-2 Hydroxy Fatty Acid Moiety of Sophorose Lipid Produced by Candida bombicola

Candida bombicola can synthesize monohydroxy fatty acid as a moiety of sophorose lipids. The hydroxy fatty acids contained in a major lactone were identified by GC-MS, after culturing with natural oils such as coconut, rapeseed, olive, and soybean oils. Hydroxy fatty acids of C18 and C16 were always synthesized, but differences were observed among the oils regarding the positions of hydroxyl groups, unsaturation, and composition of the fatty acids. A new C17 hydroxy acid was found without addition of oil.     

Cuticular lipids of adults and puparia of the Australian sheep blowfly Lucilia cuprina (Wied.)

The presence of a strong contact component in the sex and ovipositing behavior of the sheep blowfly Lucilia cuprina Wied. prompted an investigation into the chemical composition of the cuticular wax of the adult male and female flies as well as that of the blowfly puparia. Thin-layer chromatography indicated that the lipids in all the waxes examined comprise hydrocarbons, nonglyceryl esters, triglycerides, free fatty acids, and hydroxy compounds, probably diglycerides and monoglycerides. Phospholipids were not detected. Straight-and branched-chain saturated compounds, the latter often pre-dominating, are present in the hydrocarbon, free fatty acid, and ester fractions. Unsaturated molecules were absent. The hydrocarbons resemble those of the cricket to some extent, but the absence of unsaturated compounds is in striking contrast to both the cricket and the cockroach. Pheromones may be present in the low molecular weight fatty acids obtained on brief extraction of the insects.     

Isolation of lipids from photosystem I complex and its characterization with high performance liquid chromatography/electrospray ionization mass spectrometry

A method for simultaneous analysis of lipids extracted from photosystem I complex was developed with high performance liquid chromatography/electrospray ionization mass spectrometry. The photosystem I complex was firstly solubilized and separated using deoxycholate polyacrylamide gel electrophoresis method after ultrasonic treatment of the sample (leaves of pea, Pisum sativum L.). The Photosystem I complexes were electrophoretically eluted from the deoxycholate polyacrylamide gel electrophoresis bands containing them, and the electron transport activity of the eluent measured as confirmation. Lipids, which were isolated from the complex having photosystem I activity, were separated and characterized with high performance liquid chromatography/electrospray ionization mass spectrometry. Five lipids, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, phosphatidylglycerol, sulphoquinovosyldiacylglycerol and phosphaditylcholine were found combining with photosystem I complex. Different species of these lipids were found in the ESI mass spectra and the compositions of the acyl groups in them were determined.     

Ornithine-containing lipids of some Pseudomonas species

Ornithine-containing lipids purified by thin-layer chromatography were found to represent 2-15% of the total extractable cellular lipids in two or three strains each of four Pseudomonas species: P. aeruginosa, P. fluorescens, P. stutzeri and P. cepacia. The structures of the ornithine-containing lipids were elucidated by chemical analysis, thin-layer chromatography, gas-liquid chromatography, gas-liquid chromatography/mass spectrometry (electron impact or secondary ion) and infrared absorption spectroscopy. At least six molecular species of ornithine-containing lipids were present in common in all of the preparations of the four Pseudomonas species. The structure which was the most abundantly in P. fluorescens (about 60% of the total amount of the ornithine-containing lipid) was 3-hydroxyhexadecanoic acid amide-linked to ornithine and esterified to hexadecanoic acid. In addition to this structure, 3-hydroxyoctadecenoic acid amide-linked to ornithine and esterified to hexadecanoic acid was a dominant structure in the ornithine-containing lipids of P. aeruginosa, P. stutzeri or P. cepacia. In P. cepacia, another ornithine-containing lipids with a terminal polar fatty acid, 3-hydroxyhexadecanoic acid amide-linked to ornithine and esterified to 2-hydroxynonadecacyclopropanoic acid or 2-hydroxyoctadecenoic acid, was found; its content, which represented 8-11% of the total extractable cellular lipids, was higher than that of the ornithine-containing lipids with a terminal nonpolar fatty acid. These ornithine-containing lipids exhibited hemagglutinating activity. Additionally, it was very interesting that hydroxy fatty acids included in the ornithine-containing lipids were not found in the phospholipids which represented more than 80% of the total extractable cellular lipids.     

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.     

Untersuchungen zur gelpermeationschromatographischen Fraktionierung von Natrium-Ligninsulfonat

The gelpermeation chromatography of sodium ligncsulfonates with bidistilled water as an eluent is influenced by the volume expansion of the polyelectrolyte, by the negative changes of the gels, and by the inclusion effect so markedly that true conclusions on the molecular weight distribution of sodium lignosulfonate cannot be drawn. Using electrolytes as an eluent these irregularities can be avoided. Polydispersity of these macromolecules however prevents separation into discrete peaks. As a result of this behaviour changes of molecular weight distribution are only poorly recognized. Therefore, on studying biotransformation of lignosulfonic acids we purpose to use gels by which one part of the lignosulfonates is excluded, and biotransformation may be controlled by the ratio of excluded part of the lignosulfonates to that part retarded by the gels.     

Sophorose lipid production from lipidic precursors: Predictive evaluation of industrial substrates

Summary Sophorose lipids stand out as biosurfactants with a wide potential for industrial application and which can be produced in good yield from glucose and a lipidic cosubstrate.Candida bombicola CBS 6009 (ATCC 22214) was used in the present study. The influence of the lipidic cosubstrate on various aspects of production performance of these glycolipids (final concentration, yield) and on product composition (in particular, the structure of the hydroxy fatty acid vegetable and animal oils, markedly influenced product composition. In terms of production performance, the best substrates were oils or esters rich in C18:0 and C18:1 fatty acids. Optimal overall performance was obtained with esters (340 g L^–1 sophorose lipids with rapeseed esters). Conclusions drawn from the results allow predictive evaluation of lipidic industrial substrates.     

Induction of vesicle-to-micelle transition by bile salts for DOPE vesicles incorporating immunoglobulin G.

The vesicle-to-micelle transition of immunoliposomes formed by dioleoylphosphatidyl-ethanolamine (DOPE) and palmitoyl-immunoglobulin G (p-IgG) was investigated in the presence of bile salts and conjugated bile salts. Turbidity and the release of calcein from liposomes were measured as a function of the amount of bile salts added and compared with the solubilizing profiles of the salts according to the number and configurational state of hydroxy groups in the cholate. The solubilizing phenomena by bile salts conjugated with glycine or taurine were investigated in comparison with non-conjugated bile salts. The solubilizing effect of bile salts on the bilayer of immunoliposomes increased remarkably with the number of hydroxy groups, but was not influenced by the configurational state of the hydroxy group. The half-maximal concentration of bile salts, defined as the concentration giving the half-maximum turbidity of liposome solutions, decreased with hydrophobicity in the phosphatidylcholine (PC) bilayer. The increase in the hydrophobicity of bile salts induces the ability to permeabilize and solubilize phospholipid vesicles. In the case of PC or PE liposome bilayers with inserted protein, bile salts conjugated with taurine or glycine had lower hydrophobicity than non-conjugated bile salts and showed a lower half-maximal concentration. The conjugated bile salts are believed to interact with lipids and solubilize the bilayers, while the head groups of bile salts interact with the inserted protein and extract it from the lipid bilayer.     

Isolation of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine from the bull heart

A novel efficient procedure based on silica gel column chromatography has been developed for isolating chromatographically pure diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. For this purpose the lipid extract is treated with aqueous MgCl2, whereas aqueous ammonia is added to the eluent systems. To raise the yield of lipids, a fractional loading of columns with portions of lipid extracts is employed, each loading being followed by partial elution of neutral lipids with chloroform. Optimal phospholipid--silica gel ratio is 1:20 for such a procedure. The presence of cation exchange between the lipid extract and silica gel is confirmed and its influence on the efficacy of phospholipid separation is discussed.