Unsaturated Fatty Acid Moieties Research Articles

Sophorolipid production from different lipid precursors observed with LC-MS

An HPLC-MS n system was used to quantify and identify the structures of individual sophorolipid components produced in Torulopsis bombicola fermentation on glucose with or without hexadecane or soybean oil. With glucose alone, the SL production was minimal and the products were complex mixtures with mainly acidic SLs. The SLs produced with glucose plus soybean oil were also complex, containing both lactonic and acidic SLs with saturated and unsaturated C16 and C18 fatty acid moieties. The glucose plus hexadecane system gave the highest production rate and product selectivity, forming primarily two diacetylated lactonic isomers with palmitate as the fatty acid moiety. A close structure correspondence between the SL’s lipid moiety and the lipid precursor used was observed. The change of the composition of SL mixtures along batch fermentation was further examined. The concentrations of acidic SLs increased very gradually throughout the process. The production of lactonic SLs became appreciable following the addition of hexadecane or soybean oil at 24 h, and increased much more rapidly after the culture reached the stationary phase. The combined percentage of the main lactonic SLs leveled off at ∼ 80% for the hexadecane system and ∼ 50% for the soybean oil system. The yields of crude SLs were 0.84, 0.20, and 0.03 g per gram of hexadecane, soybean oil, and glucose consumed during the SL production phase. Hexadecane is thus a more efficient second C-source for sophorolipid production.

A novel analytical method to detect adulteration of virgin olive oil by other oils

AbstractThe present study focuses on the olefinic region of the 13C nuclear magnetic resonance (13C NMR) spectrum of virgin olive oil which shows 12 peaks resonating between 127.5 and 130 ppm. These peaks are assigned to the most abundant unsaturated fatty acid moieties of the olive oil, oleic and linoleic acids, which are present in α and β positions of the glycerol backbone. With the use of an internal reference pyrazine, the 12 peaks were integrated and their areas were expressed in mmol/g of virgin olive oil. The intensities of the 12 observed peaks were affected when an authentic virgin olive oil was mixed with a seed oil. This observation was used to develop a semiquantitative method to detect adulteration of virgin olive oil by other oils based on 13C NMR spectroscopy.

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.

Isolation and Characterization of Wax Esters in Fennel and Caraway Seed Oils by SPE-GC

Summary A rapid method for the isolation and quantitative determination of wax esters in vegetable oils was developed. For the first time wax esters in oils were separated from the triglyceride matrix by means of solid-phase extraction, which allows rapid sample preparation in parallel and therefore a high sample throughput. The thus obtained wax ester fractions of fennel and caraway seed oils were analyzed by high temperature gas chromatography. GC-MS analyses were carried out using electron impact ionization in order to characterize the wax ester fraction. With respect to the results of the GC-MS analyses different isomers of saturated wax esters with the same carbon number were observed. Additional monounsaturated wax esters with an unsaturated fatty acid moiety were identified.

Isolation and Characterization of Wax Esters in Fennel and Caraway Seed Oils by SPE-GC

A rapid method for the isolation and quantitative determination of wax esters in vegetable oils was developed. For the first time wax esters in oils were separated from the triglyceride matrix by means of solid-phase extraction, which allows rapid sample preparation in parallel and therefore a high sample throughput. The thus obtained wax ester fractions of fennel and caraway seed oils were analyzed by high temperature gas chromatography. GC-MS analyses were carried out using electron impact ionization in order to characterize the wax ester fraction. With respect to the results of the GC-MS analyses different isomers of saturated wax esters with the same carbon number were observed. Additional monounsaturated wax esters with an unsaturated fatty acid moiety were identified.

Insights into the molecular basis for fatty acyl specificities of lipases from Geotrichum candidum and Candida rugosa

Despite immense progress in our comprehension of lipase structure and function during the past decade, the basis for lipase acyl specificities has remained poorly understood. This review summarizes some recent advances in the understanding at the molecular-level of substrate acyl recognition by two members in a group of large ( M w∼60 kDa) microbial lipases. Two aspects of acyl specificity will be focused upon. (i) The unique preference of a fungal Geotrichum candidum lipase for long-chain cis (Δ-9) unsaturated fatty acid moieties in the substrate. Mutational analysis of this lipase identified residues essential for its anomalous acyl preference. This information highlighted for the first time parts in the lipase molecule involved in substrate acyl differentiation. These results are discussed in the context of the 3D-structure of a G. candidum lipase isoenzyme and structures of the related Candida rugosa lipase in complex with inhibitors. (ii) The mechanism by which the yeast C. rugosa lipase discriminates between enantiomers of a substrate with a chiral acyl moiety. Molecular modeling in combination with substrate engineering and kinetic analyses, identified two alternative substrate binding modes. This allowed for the proposal of a molecular mechanism explaining how long-chain alcohols can act as enantioselective inhibitors of this enzyme. A picture is thus beginning to emerge of the interplay between lipase structure and fatty acyl specificity.

Structures and Physical Properties of Crystalline Fatty Acids and Triacylglycerols

Recent studies of the structures and physical properties of crystalline fatty acids and triacylglycerols are reviewed, in an attempt to construct a molecular-level understanding of crystal structures and mixing phase behavior of the materials examined. In particular, polymorphism and mixing behavior of various cis-and trans-monounsaturated fatty acids and triacylglycerols have been discussed. One may draw a conclusion that acyl-acyl interactions through the unsaturated and saturated fatty acid moieties are critically important in determining the phase behavior of the mixture systems of the triacylglycerols, forming molecular compounds or supramolecules in a special case.

Phospholipids of Rhizobium contain nodE-determined highly unsaturated fatty acid moieties

In Rhizobium leguminosarum the nodABC and nod-FEL operons are involved in the production of lipooligosaccharide signals, which mediate host specificity. A nodE-determined highly unsaturated C18:4 fatty acid (trans-2,trans-4,trans-6,cis-11-octadecatetraenoic acid) is essential for the ability of the signals to induce nodule primordia (Spaink, H. P., Sheeley, D. M., van Brussel, A. A. N., Glushka, J., York, W.S., Tak, T., Geiger, O., Kennedy, E. P., Reinhold, V. N., and Lugtenberg, B. J. J. (1991) Nature 354, 125-130) and preinfection thread structures (van Brussel, A. A. N., Bakhuizen, R., van Spronsen, P. C., Spaink, H. P., Tak, T., Lugtenberg, B. J. J., and Kijne, J. W. (1992) Science 257, 70-72) on the host plant Vicia sativa. We presently focus on the question of how these lipo-oligosaccharide signals are synthesized in Rhizobium. Here we show that after the induction of the nodFE genes, even in the absence of the nodABC genes, the trans-2,trans-4,trans-6,cis-11-octadecatetraenoic acid, which has a characteristic absorbance maximum of 303 nm, is synthesized; this shows that the biosynthesis of the unusual fatty acid is not dependent on the synthesis of the lipooligosaccharides. This finding also suggests that the biosynthesis of the unusual fatty acid is completed before it is linked to the sugar backbone of the lipooligosaccharide. In an attempt to identify the lipid fraction with which the unusual C18:4 fatty acid is associated, we found that it is linked to the sn-2 position of the phospholipids. Even when lipooligosaccharide signals are produced in a wild type Rhizobium cell, a fraction of the unusual fatty acid is still bound to all major phospholipids. These findings offer interesting possibilities. 1) The phospholipids might be biosynthetic intermediates for the synthesis of lipooligosaccharide signals, and 2) phospholipids, containing nodFE-derived fatty acids, might have a signal function of their own.

In vitro oxygenation of soybean biomembranes by lipoxygenase-2

The ability of soybean lipoxygenases-1 and -2 to oxygenate biomembranes isolated from soybean seedlings has been investigated. Constituents of the lipid bilayer were analyzed by means of reversed phase and chiral phase high performance liquid chromatography, gas chromatography/mass spectrometry, high performance thin layer chromatography and uv spectroscopy. Evidence is presented that soybean lipoxygenase-2, at variance with the type-1 enzyme, oxygenates the esterified unsaturated fatty acid moieties in biomembranes, whereas membrane-embedded free unsaturated fatty acid moieties were not a suitable substrate for either isoenzyme. The oxygenation products derived from the biomembranes were the 9- and 13-hydroperoxides of linoleic acid residues, in a molar ratio of 1.0 to 1.7, and the 9- and 13-hydroperoxides of α-linolenic acid residues, in a molar ratio of 1.0 to 0.1. The R S ratios of 13-hydroperoxy-9 Z,11 E-octadecadienoic acid and 9-hydroperoxy-10 E,12 Z,15 Z-octadecatrienoic acid were found to be 0.5 and 25.0, respectively. These stereospecificity values were much higher than those of hydroperoxides isolated after incubation of lipoxygenase-2 with non-membraneous fatty acids or their methyl esters. The hydroperoxy fatty acids produced were distributed in neutral lipids and phospholipids isolated from soybean membranes, the former being oxidized to a larger extent. Furthermore, both intracellular and plasma membranes were substrates for the enzymic oxygenation, with a preference for those of chloroplasts followed by those of Golgi apparatus, endoplasmic reticulum, plasma membrane and mitochondria. These data point towards a different action of the two lipoxygenases in soybean cells. We suggest that the type-2 enzyme plays a role in the in vivo remodelling of biomembranes. The physiological relevance of these findings is discussed.

Synthesis of a highly unsaturated fatty acid moiety of lipo oligosaccharides determining host‐specificity in rhizobium

AbstractThe phosphonate ethyl 6‐(diethylphosphono)‐2E,4E‐hexadienoic acid is a suitable synthon for the highly stereoselective preparation of 2E,4E,6E,11Z‐octadecatetraenoic acid and its 2E,4E,6E‐ analog.

A novel highly unsaturated fatty acid moiety of lipo-oligosaccharide signals determines host specificity of Rhizobium

In Rhizobium leguminosarum biovar viciae, the nodABC and nodFEL operons are involved in the production of lipo-oligosaccharide signals which mediate host specificity. The structure of these metabolites and those produced in nod mutants links the nodE and nodL genes to specific chemical features of the signal molecules. A nodE-determined, highly unsaturated fatty acid and a nodL-determined O-acetyl substituent are essential for the ability of the signals to induce nodule meristems on the host plant Vicia sativa.

Neurite-Promoting Activities of Phosphatidylinositol and Other Lipids on Fetal Rat Septal Neurons in Culture

Neurite-promoting activities of lipids were assessed using serum-free cultures of fetal rat septal neurons. The most potent one was phosphatidylinositol (PI), followed by PI 4-phosphate, phosphatidylserine, sphingomyelin, and phosphatidylcholine. The EC50 value for PI was 1.5 micrograms/ml (1.8 microM), and activity was maximal at 4 micrograms/ml (56% of total cells had neurites after 24 h). Cerebroside, sulfatide, and di- and triacylglycerols showed relatively low activities. Synthetic dipalmitoyl phosphatidylcholine was also active, with a maximal activity (47%) at 100 micrograms/ml, a finding implying that the unsaturated fatty acid moiety is not released and further used as substrate for the arachidonic acid cascade. Lysophospholipids, phosphatidylglycerol, and cardiolipin were rather cytotoxic and lacked activity, an observation suggesting that membrane perturbation is not responsible for the neurite-promoting activity. Treatment with a protein kinase C inhibitor, H-7, or an Na+,K(+)-ATPase inhibitor, ouabain, inhibited the PI-induced neurite outgrowth, but the cyclic AMP- and cyclic GMP-dependent protein kinase inhibitor HA1004 did not inhibit this activity, a result indicating that multiple elements (protein kinase C and Na+,K(+)-ATPase) are involved in the induction of neurites. Because phospholipids can be provided either as lipid vesicles or as lipoproteins produced by macrophages at regeneration sites, they may play an important role in the regeneration of certain populations of neurons.

The mitogenic activities of phosphatidate are acyl-chain-length dependent and calcium independent in C3H/10T1/2 cells.

Phosphatidates (PA or phosphatidic acid) were shown to have mitogenic properties, including the stimulation of DNA synthesis and calcium mobilization in C3H/10T1/2 cells. Their continuous presence for a minimum of 7 h induced DNA synthesis with kinetics similar to that observed when 10% fetal bovine serum was used as a mitogen. PAs with long chain saturated fatty acid moieties were more mitogenic, in a dose-dependent fashion, than PAs with short saturated or unsaturated fatty acid moieties. When compared with lysostearoyl-PA (LSPA), distearoyl-PA (DSPA) was as potent with respect to the induction of DNA synthesis. Lysooleoyl-PA (LOPA) was slightly more potent than dioleoyl-PA (DOPA), but much weaker than DSPA and LSPA. Preincubation with dilauroyl-PA (DLPA) reduces the mitogenic effect of DSPA by 85%. The pattern of mitogenic inhibition suggests that a chain-length-independent, yet PA-specific, mechanism is involved. Both DSPA and DLPA are equally taken up by the cells after 30 min. LOPA, but not LSPA, produced a large calcium transient (1.3 microM), which we found to be derived from intracellular sources. DSPA, the most mitogenic PA tested, produced a weaker transient (0.6 microM). Interestingly, LSPA did not produce any detectable calcium transient. These results suggest that the chain-length-specific step in the signaling mechanism of PA occurs after the initial chain-length-independent partitioning and/or binding to the membrane and that the induction of DNA synthesis is not related to the observed calcium transients.

Membrane stabilizing effect of vitamin E: Existence of a hydrogen bond between .ALPHA.-tocopherol and phospholipids in bilayer liposomes.

In order to elucidate the existence of a hydrogen bond between alpha-tocopherol and phospholipids in bilayer liposomes, the effects of the presence or absence of alpha-tocopherol or its acetate on either the permeability of liposomes to chromate ions or the fluidity of liposomes composed of the different kinds of lecithin [egg yolk phosphatidylcholine (EPC), 1-oleoyl-2-palmitoylphosphatidylcholine (OPPC), or 1-O-oleyl-2-O-palmitoylphosphatidylcholine (OPPCE)] were examined. From the results obtained, it was concluded that the hydroxyl group of alpha-tocopherol is hydrogen bonded to the carbonyl group of a fatty acid ester in the phospholipids of bilayer liposomes in order to retain alpha-tocopherol molecule in the space close to the surface of membranes formed by the unsaturated fatty acid moiety of phospholipids.

Reassessment of the role of phospholipids in sexual reproduction by sterol-auxotrophic fungi.

Several genera of oomycete fungi which are incapable of de novo sterol synthesis do not require these compounds for vegetative growth. The requirement for an exogenous source of sterols for sexual reproduction by several members of the Pythiaceae has been questioned by reports of apparent induction and maturation of oospores on defined media supplemented with phospholipids in the absence of sterols. A more detailed examination of this phenomenon suggested that trace levels of sterols in the inoculum of some pythiaceous fungi act synergistically with phospholipid medium supplements containing unsaturated fatty acid moieties to induce oosporogenesis. Phospholipid analysis of one species, Pythium ultimum, suggested that only the fatty acid portion of the exogenous phospholipid is taken up by the fungus. Enrichment of the phospholipid fraction of total cell lipid of P. ultimum with unsaturated fatty acids promoted oospore induction, and enhanced levels of unsaturated fatty acids in the neutral lipid fraction increased oospore viability. For some pythiaceous fungi, the levels of sterols required for the maturation of oospores with appropriate phospholipid medium supplementation suggest that these compounds are necessary only for the sparking and critical domain roles previously described in other fungi.

Effects of phosphatidylcholines containing furan fatty acid on oxidation in multilamellar liposomes.

Several phosphatidylcholines (PCs) containing furan fatty acid (10, 13-epoxy-11, 12-dimethyloctadeca-10, 12-dienoic acid, F acid) were synthesized, and their antioxidant activities were studied in the oxidation of soybean PC. The rate of oxidation in soybean PC liposomes initiated with 2, 2'-azobis(2, 4-dimethylvaleronitrile) (AMVN), a lipophilic azoinitiator, was measured by following the oxygen uptake, the accumulation of conjugated diene, and the disappearance of unsaturated fatty acid moieties. These PCs containing F acid were found to retard the rate of oxidation in soybean PC liposomes, and the retarding effect increased in the following order : 1/2 mol of di-furanoacyl PC (di-F PC)<1 mol of 1-furanoacyl-2-linoleoyl PC (F-18 : 2 PC)<1 mol of 1-furanoacyl-2-arachidonyl PC (F-20 : 4 PC)<1 mol of di-F PC (1 : 1 : 1 : 2, as molar ratios of F acid residue). This is the first report showing that the naturally occurring F acid has a retarding effect on lipid peroxidation.

Interactions of lipids with peripheral-type benzodiazepine receptors

Peripheral-type benzodiazepine receptors (PBRs) are present at high densities in the rat kidney distal tubule. [ 3H]RO 5-4864 binding to PBRs in kidney membranes is inhibited by several unidentified low molecular weight hydrophobic compounds in urine and serum. We tested representative hydrophobic compounds from several lipid classes for ability to inhibit binding to rat kidney PBRs of two high affinity ligands, [ 3H]RO 5-4864 and [ 3H]PK 11195. Unsaturated fatty acids and alcohols inhibited [ 3H]RO 5-4864 binding with half-maximal inhibition occurring at 3 × 10 −6 M to 10 −4 M. Inhibitory potency increased with the degree of unsaturation. Phospholipids inhibited [ 3H]RO 5-4864 in the same concentration range, with inhibitory potency in this case dependent both upon an unsaturated fatty acid moiety and upon the polar head group. Phosphatidylethanolamine was the most potent phospholipid tested ( ic 50 = 2 × 10 −6M), whereas phosphatidylcholine was not inhibitory. Although phospholipids inhibited both [ 3H]Ro 5-4864 and [ 3H]PK 11195 binding equally, unsaturated fatty acids had a much greater inhibitory effect upon [ 3H]RO 5-4864 than upon [ 3H]PK 11195 binding. Similar effects were obtained with digitonin-solubilized PBRs. These data demonstrate that in our experiments PBR binding was inhibited by specific lipids and that binding of proposed agonist (RO 5-4864) and antagonist (PK 11195) ligands was differentially affected by unsaturated fatty acids.

Characterization of glutathione-dependent inhibition of lipid peroxidation of isolated rat liver nuclei

Glutathione (GSH) is known to play an important role in protecting cells against oxidative stress. The present study was undertaken to assess the ability of GSH to protect isolated rat liver nuclei against NADPH-induced peroxidation. Nuclei were isolated from rat liver homogenates by discontinuous sucrose gradient centrifugation, and lipid peroxidation was induced by 1.7 m m ADP, 0.11 m m EDTA, 0.1 m m FeCl 3, and either 1 m m NADPH or 0.5 m m ascorbate. The amount of lipid peroxidation was determined by measuring the formation of thiobarbituric acid-reactive products and the disappearance of lipid unsaturated fatty acid moieties. The addition of GSH (0.1 to 1.0 m m) produced a concentration-dependent lag period prior to the onset of lipid peroxidation. This GSH-induced lag period was abolished by pretreatment of nuclei with trypsin, thiol modifying reagents, disulfides, or heating nuclei at 60 °C for 15 min. Nuclei which were incubated with GSH also catalyzed the conversion of cumene hydroperoxide to cumyl alcohol. Similarly, this activity was also inhibited by thiol modifying reagents, disulfides, and heating nuclei at 60 °C for 15 min. The data suggest that a GSH-dependent peroxidase activity is associated with rat liver nuclear membranes which are capable of inhibiting lipid peroxidation.

Glidobactins A, B and C, new antitumor antibiotics. II. Structure elucidation.

The structures of new antitumor antibiotics, glidobactins A (Ia), B (Ib) and C (Ic) were elucidated by a combination of chemical and enzymatic degradations and spectral analyses. They have in common a cyclized tripeptide nucleus composed of L-threonine, 4(S)-amino-2(E)-pentenoic acid and erythro-4-hydroxy-L-lysine, and differ from each other in the unsaturated fatty acid moiety attached to the peptide.

An ESR study on lipid peroxidation process. Formation of hydrogen atoms and hydroxyl radicals

Lipid peroxidation process was studied by the combination of ESR spectroscopy and spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide. Autoxidation of egg lecithin phosphatidylcholine dispersed in water was found to produce hydrogen atoms and hydroxyl radicals. It was also found that for producing such reactive species, unsaturated fatty acid moieties are needed. The ESR spectrum obtained from this model system was identical with that from body fluid such as serum, indicating that hydrogen atoms and hydroxyl radicals could be generated in living bodies during the course of lipid peroxidation.