Long-chain Fatty Acid Esters Research Articles

The potential role of adenosine in the pathophysiology of the insulin resistance syndrome

An increased intracellular availability of the co-enzyme A esters of long-chain fatty acids is thought to underlie many aspects of the insulin resistance syndrome. However, the cause of clustering of a hyperdynamic circulation, sympathetic activation, hypertension, hyperuricaemia, and a raised haematocrit in the insulin resistance syndrome remains to be elucidated. We propose a mechanism that expands the etiological role of long-chain fatty acids. By inhibiting adenine nucleotide translocators, elevated intracellular concentrations of the co-enzyme A esters of long-chain fatty acids impair mitochondrial oxidative phosphorylation. This is expected to result in a chronic systemic increase in extracellular adenosine concentrations. As adenosine stimulates the sympathetic nervous system, induces systemic vasodilatation, stimulates erythropoiesis, and induces renal vasoconstriction with renal sodium retention, increased extracellular ADO concentrations may be the common denominator explaining the above-mentioned and still unexplained phenomena associated with the insulin resistance syndrome. Along the same lines, hyperuricaemia can be explained by the fact that adenosine is broken down to urate and because of increased renal urate retention.

Cooperation of liver cells in health and disease.

Cooperation of liver cells in health and disease.

Purification of a jojoba embryo fatty acyl-coenzyme A reductase and expression of its cDNA in high erucic acid rapeseed.

The jojoba (Simmondsia chinensis) plant produces esters of long-chain alcohols and fatty acids (waxes) as a seed lipid energy reserve. This is in contrast to the triglycerides found in seeds of other plants. We purified an alcohol-forming fatty acyl-coenzyme A reductase (FAR) from developing embryos and cloned the cDNA encoding the enzyme. Expression of a cDNA in Escherichia coli confers FAR activity upon those cells and results in the accumulation of fatty alcohols. The FAR sequence shows significant homology to an Arabidopsis protein of unknown function that is essential for pollen development. When the jojoba FAR cDNA is expressed in embryos of Brassica napus, long-chain alcohols can be detected in transmethylated seed oils. Resynthesis of the gene to reduce its A plus T content resulted in increased levels of alcohol production. In addition to free alcohols, novel wax esters were detected in the transgenic seed oils. In vitro assays revealed that B. napus embryos have an endogenous fatty acyl-coenzyme A: fatty alcohol acyl-transferase activity that could account for this wax synthesis. Thus, introduction of a single cDNA into B. napus results in a redirection of a portion of seed oil synthesis from triglycerides to waxes.

Long-chain alkanediols, ketoaldehydes, ketoalcohols and ketoalkyl esters in the cuticular waxes of Osmunda regalis fronds

In the frond waxes of Osmunda regalis four homologous series of previously unknown compounds were detected. In order to assign their structures various derivatives were prepared and analyzed by GC-MS. The wax components were identified as C 28 to C 32 alkanediols, C 26 to C 36 ketoalcohols, C 28 to C 36 ketoaldehydes and long-chain fatty acid esters of C 28 to C 36 ketoalcohols. All fractions showed strong prevalence of even numbered homologues with a maximum for C 30. Isomer distributions were in all cases dominated by 1,9, 1,11-, 1,13- and 1,15-compounds, their relative abundances shifting towards the latter with increasing homologue chain length. A total of 77 (69 novel) compounds were identified, partially as minor components in unresolved isomer mixtures.

Localization of adipocyte long-chain fatty acyl-CoA synthetase at the plasma membrane

Long-chain fatty acyl-CoA synthetase (FACS) catalyzes esterification of long-chain fatty acids (LCFAs) with coenzyme A (CoA), the first step in fatty acid metabolism. FACS has been shown to play a role in LCFA import into bacteria and implicated to function in mammalian cell LCFA import. In the present study, we demonstrate that FACS overexpression in fibroblasts increases LCFA uptake, and overexpression of both FACS and the fatty acid transport protein (FATP) have synergistic effects on LCFA uptake. To explore how FACS contributes to LCFA import, we examined the subcellular location of this enzyme in 3T3-L1 adipocytes which natively express this protein and which efficiently take up LCFAs. We demonstrate for the first time that FACS is an integral membrane protein. Subcellular fractionation of adipocytes by differential density centrifugation reveals immunoreactive and enzymatically active FACS in several membrane fractions, including the plasma membrane. Immunofluorescence studies on adipocyte plasma membrane lawns confirm that FACS resides at the plasma membrane of adipocytes, where it co-distributes with FATP. Taken together, our data support a model in which imported LCFAs are immediately esterified at the plasma membrane upon uptake, and in which FATP and FACS function coordinately to facilitate LCFA movement across the plasma membrane of mammalian cells.—Gargiulo, C. E., S. M. Stuhlsatz-Krouper, and J. E. Schaffer. Localization of adipocyte long-chain fatty acyl-CoA synthetase at the plasma membrane. J. Lipid Res. 1999. 40: 881–892.

Charge-remote molecular hydrogen removal in protonated and alkali-cationized long-chain fatty acid esters upon cesium ion bombardment

The mass spectral behavior of cationized saturated fatty acid derivatives has been studied in order to gain insight into the loss of molecular hydrogen during cesium ion bombardment. It is shown that molecular hydrogen and hydrogen radical loss occurs for protonated and alkali (Li +/Na +)-cationized fatty acid methyl esters as well as for protonated acylcarnitines and that molecular hydrogen loss is dependent upon the acyl chain length. Investigation of ion structures by collision-induced dissociation tandem mass spectrometry indicates that in the case of alkali-cationization a hydrogen radical is lost from all positions of the saturated acyl chain, whereas in the case of protonated molecules a hydrogen radical is predominantly eliminated from the protonated ester group. For the primary reaction of the dehydrogenation chemistry, an ion-beam-induced excitation of a cationized molecule is proposed, yielding a diradical species that gives rise to an intermolecular reaction with a neutral analyte molecule. Subsequent cationization of the neutral [M–H 2] species formed in this primary reaction leads to the formation of the satellite ion at m/ z values 2 u lower compared to the cationized molecule.

High-performance liquid chromatography and laser desorption/ionization mass spectrometry of retinyl esters.

Laser desorption/ionization mass spectrometry (LDI-MS) at 337 nm laser wavelength was used to analyze retinol and several long-chain fatty acid esters of retinol. Employing this ionization technique helped to overcome the inherent problems resulting from thermal instability of retinyl esters which render this group of compounds rather difficult for standard ionization techniques. Mass spectra were recorded with a linear time-of-flight instrument in positive ion mode. Under these conditions, retinyl esters formed radical molecular ions (M.+) and in addition fragmented by elimination of the fatty acyl chain to uniformly form a peak at m/z = 269 u. The elimination of carbon dioxide was also observed in the spectra. The method is suitable to identify specific retinyl esters in complex mixtures of these compounds. A gradient reversed-phase high-performance liquid chromatography (HPLC) is described for the separation of retinol and 15 related fatty acid esters within 28 min. HPLC was applied to separate retinyl esters from rat liver extracts. The LDI mass spectrum of the collected HPLC fraction of rat liver extract showed the molecular parent ions of retinyl myristate, pentadecanoate, palmitoleate, palmitate, heptadecanoate, linoleate, oleate, stearate, and 3,4-didehydroretinyl palmitate. LDI-MS was found to be more appropriate than matrix-assisted laser desorption/ionization for the described analytical task.

Purification and characterization of rat liver microsomal fatty acid ethyl and 2-chloroethyl ester synthase and their relationship with carboxylesterase (pI 6.1).

Previous studies have shown that fatty acid ethyl ester synthase (FAEES) which catalyzes the formation of ethyl or 2-chloroethyl esters of long-chain fatty acids is localized in the microsomal fraction of rat liver. A recent study suggests that rat adipose tissue FAEES is similar to rat liver microsomal carboxylesterase (CE) [Tsujita and Okuda (1992) J. Biol. Chem. 267, 23489-23494]. Since the interrelationships among FAEES, 2-chloroethyl ester synthase (FACEES), and cholesterol esterase (ChE) are also not clear at present, we purified and characterized FAEES and FACEES from rat hepatic microsomes and studied their functional and structural relationships with CE and ChE. The results of these studies showed that CE, FAEES, and FACEES activities copurified during each step of purification. Although gel-filtration column chromatography of DEAE-Sephacel purified microsomal protein resolved into two peaks with an estimated molecular weight of 180 (major) and 60 kDa (minor, this paper describes characterization of only the 180 kDa protein. CE, FAEES, and FACEES activities associated with homogeneous 180 kDa protein could be inhibited by a beta-esterase inhibitor (diisopropyl fluorophosphate) in an identical manner. This protein, however, showed only the hydrolytic activity, but not the synthetic activity for cholesterol oleate, indicating that it is distinct from ChE. The purified protein could be immunoprecipitated with the antibodies raised against rat adipose tissue FAEES, but not with antibodies against rat pancreatic ChE, demonstrating again that the purified protein is distinct from ChE. A single band corresponding to 60 kDa upon SDS-PAGE, under reduced denaturing conditions, indicates that the purified protein is a trimer. N-terminal amino acid sequence of the first 27 residues were identical to that of rat hepatic microsomal CE [Robbi et al. (1990) Biochem. J., 451-458] which suggests structural similarity of the purified protein with rat hepatic microsomal CE. Therefore, the functional and structural properties of the purified protein demonstrate that FAEES, FACEES, as well as CE activities are expressed by the same protein, purified in this study, which exists as a trimer (180 kDa) and is involved in biosynthesis of long-chain fatty acid esters of xenobiotic alcohols. Further studies on purification and characterization of the enzymes responsible for the esterification of xenobiotic alcohols with endogenous fatty acids from various target organs need to be conducted to determine their functional and structural interrelationships. Inhibition and induction studies of these enzyme(s) and the extent of observed toxicity could be important in understanding their role in etiology of chronic diseases induced by alcohol abuse.

Effects of Pressure on the Esterification of Long-chain Fatty Acids.

The esterification of long-chain fatty acids such as lauric, stearic, oleic, elaidic, and linoleic acids in excess alcohol was accelerated under high pressure. Activation volume in the esterification of lauric acid with methyl, ethyl, i-propyl, n-butyl, i-butyl, and s-butyl alcohols was from -13-21 cm3/mol. The esterification of unsaturated fatty oleic, elaidic, and linoleic acids with methyl alcohol was accelerated by greater compression and activation volume ranged from -27-33 cm3/mol. With dichloromethane as solvent in the esterification of long-chain fatty acids with methyl alcohol, the reactions were greatly accelerated at high pressure with apparent activation volume from 0.4 MPa to 200 MPa and as much as -80 cm3/mol. The effects of pressure on reactions in dichloromethane were virtually the same in all cases except for the reaction of linoleic acid.

Waxes of the social spiderAnelosimus eximius(Araneae, Theridiidae): Abundance of noveln-propyl esters of long-chain methyl-branched fatty acids

The pentane extract of the social spider, Anelosimus eximius (Araneae, Theridiidae), contains hydrocarbons, fatty acids and their methyl esters, and a series of novel propyl esters of long-chain methyl-branched fatty acids. The propyl esters comprise almost three-fourths of the extract and consist predominantly of odd-numbered carbon chain components. Mass spectrometric analyses of the propyl esters, their methyl esters and cyanide derivatives showed that mono-, di- and trimethyl branched components with methyl branches on even numbered carbons predominate. The major components are propyl 4,20- and 4,30-dimethylhentriacontanoate and propyl 6,20- and 6,30-trimethylhentriacontanoate. The hydrocarbon fraction consists of n-, monomethyl- and dimethylalkanes, containing a relatively high proportion of even-numbered carbon chain components. The abundance of even-numbered carbon chain length alkanes and odd-numbered carbon chain length fatty acyl groups, along with abundant methyl-branches suggest that the propionyl-CoA and its carboxylated product, methylmalonyl-CoA, play important roles in the biosynthesis of these unique waxes. Arch. Insect Biochem. Physiol. 36:295–314, 1997. © 1997 Wiley-Liss, Inc.

Purification and some properties of a thermostable acid esterase from Acidiphilium sp. AIU 409.

Extracellular and cell-bound esterases produced by Acidiphilium sp. AIU 409 were homogeneously purified from culture broth and cells, respectively, and some properties were investigated. Both esterases more rapidly hydrolyzed p-nitrophenyl acyl esters containing long-chain fatty acids from C 8:0 to C 18:0 than those containing short-chain fatty acids from C 2:0 to C 6:0. The Km values for p-nitrophenyl long-chain fatty acid esters from C 8:0 to C 18:0 were approximately 1.3-1.5 mM. The enzymes were stable at 50 degrees C for 2 days between pH 3.0 and 6.5, and optimum pH and temperature were 5.0 and 70 degrees C, respectively. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride and SDS. The molecular mass of both enzymes was estimated to be approximately 64 kDa by SDS-PAGE. The 23 amino acid sequence from the NH(2)-terminus was also the same in both enzymes. These results suggest that extracellular esterase might be composed of the same components as cell-bound esterase.

The measurement of temperature with electron paramagnetic resonance spectroscopy.

An electron paramagnetic resonance (EPR) technique, potentially suitable for in vivo temperature measurements, has been developed based on the temperature response of nitroxide stable free radicals. The response has been substantially enhanced by encapsulating the nitroxide in a medium of a fatty acid mixture inside a proteinaceous microsphere. The mixture underwent a phase transition in the temperature range required by the application. The phase change dramatically altered the shape of the EPR spectrum, providing a highly temperature sensitive signal. Using the nitroxide dissolved in a cholesterol and a long-chain fatty acid ester, we developed a mixture which provides a peakheight ratio change from 3.32 to 2.11, with a standard deviation of 0.04, for a temperature change typical in biological and medical applications, from 38 to 48 degrees C. This translated to an average temperature resolution of 0.2 degree C for our experimental system. The average diameter of the nitroxide mixture-filled microspheres was approximately 2 microns. Therefore, they are compatible with in vivo studies where the microspheres could be injected into the microvasculature having a minimum vessel diameter of the order of 8 microns. This temperature measuring method has various potential clinical applications, especially in monitoring and optimizing the treatment of cancer with hyperthermia. However, several problems regarding temperature and spatial resolution need to be resolved before this technique can be successfully used to monitor temperatures in vivo.

Volatiles in Distillates of Cider Aged in American Oak Wood

A fast, reliable method is described for determining volatile components in distillates of different kinds of cider aged in oak wood. During aging, a significant increase was detected in the concentration of diethyl butanedioate, ethyl 3-methylbutanoate, esters of long-chain fatty acids, and 1-hexanol; in contrast, the acetate esters and long-chain fatty acids decreased. When cider made with traditional technology was used as raw material, spirits with higher concentrations of decanoic and dodecanoic ethyl esters and long-chain fatty acids were obtained. When cluster and factor analyses were employed, “natural” groupings among the spirits studied were obtained on the basis of the raw material used and aging time. Keywords: Volatile compounds; cider brandy; GC−MS

Synthesis of (9R)- and (9S)-10-fluorodecan-9-olide (fluoro-phoracantholide I) first lipase-catalyzed enantioselective esterification of β-fluoroalcohols

Synthesis of both enantiomers of fluoro-phoracantholide I, namely (9 S )-10-fluorodecan-9-olide ( (9S)-(−)- 1 and (9 R )-10-fluorodecan-9-olide (9R)-(+)- 1 , has been achieved through lipase-catalyzed enantioselective acetylation of methyl 10-fluoro-9-hydroxydecanoate ( 2a ) with acetic anhydride in the presence of Lipase Amano PS ( Pseudomonas cepacia ). The higher homologue, methyl 11-fluoro-10-hydroxyundecanoate ( 2b ) has also been resolved. These enzymatic resolutions by acetylation of ω-fluoro-(ω-1)-hydroxyalkanoic acid esters of longchain fatty acids represent the first lipase-catalyzed enantioselective esterifications of β-fluoroalcohols. Synthesis of both enantiomers of Fluoro-Phoracantholide I ( 1 ) key-step: enantioselective acetylation of β-fluoroalcohols with acetic anhydride and lipase of Pseudomonas cepacia .

Thermally Induced Domain Growth in Fatty Acid Ester Monolayers

Monolayers of long-chain fatty acid esters evolve irregularly shaped morphological structures depending on the existing conditions of spreading. A reproducible evolution of defined circular condensed phase domains is achieved by a heating-recooling cycle at a fixed area per molecule in the surface pressure region of about 0 mN m -1. The mechanism of domain growth is qualitatively related to a nucleation-growth mechanism. Besides the condensed phase domains that coexist with the fluid phase of low density, random cellular structures appear in it which evolve to a two-dimensional network of boundary lines under the influence of the temperature gradient. The cellular patterns within this phase of low density are accounted for by dissipative structures induced by temperature gradients.

Long-chain fatty acid esters of some norditerpenoid alkaloids.

Several C-8 long-chain fatty acid esters of aconitine-type norditerpenoid alkaloids namely, aconitine, delphinine, delphisine, falconerine-8-acetate, mesaconitine, and pseudaconitine bearing a C-8 acetyl group, have been prepared. Preparation was achieved by heating the alkaloids with the neat fatty acid under vacuum. All the compounds have been fully characterized by physical and spectroscopic data. Identification and characterization of reaction byproducts are also included.

GC-mass spectrometry of refuse-derived composts

Refuse-derived compost (RDC) was produced by the mechanical separation of organic matter from mixed refuse followed by a thermophilic composting phase. At various stages of the process, dichloromethane extracts of the compost were obtained and constituents identified by gas chromatography-mass spectrometry (GC-MS). Unstable compost contained numerous substances of uncertain origin such as alkyl and benzyl phthalates and long-chain fatty acid esters. Stable compost contained very few extractable organic constituents.

Cloning of a rat adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during preadipocyte differentiation. Homology with human CD36.

A cDNA for an adipocyte membrane protein, implicated in the transport of long-chain fatty acids, was isolated by screening with a synthetic oligonucleotide derived from the amino terminal sequence of the protein. The 88-kDa adipocyte membrane protein was previously identified by covalent labeling with N-sulfosuccinimidyl esters of long-chain fatty acids which irreversibly inhibited fatty acid transport by 75% (Harmon, C. M., and Abumrad, N.A. (1993) J. Membr. Biol. 124, 261-268). The cDNA (FAT, 2432 base pairs (bp)) contained 70 bp of 5'-untranslated sequence, an open reading frame encoding a 472-amino acid protein with a predicted molecular mass of 52466, and 940 bp of 3'-untranslated sequence with two polyadenylation signal sequences but with no polyadenylation tail. The deduced protein sequence predicted two transmembrane segments and 10 potential N-linked glycosylation sites. Extensive glycosylation most likely explains why the molecular mass of the isolated protein (88 kDa) is different from that deduced from the cDNA sequence (53 kDa). The sequence of FAT is 85% homologous with that of glycoprotein IV (CD36) identified in human platelets and in lactating mammary epithelium. Consistent with this, a polyclonal antibody against CD36 reacted with adipocyte plasma membranes and detected a single band at 88 kDa. Northern blot analysis of RNA obtained from rat adipose tissue and probed with the cDNA identified two major transcripts of 4.8 and 2.9 kilobases which were abundant in heart, intestine, fat, muscle, and testis. The mRNAs were not detectable in cultured adipose cell lines (Ob1771, 3T3F442A) at the fibroblastic stage but were strongly induced during the differentiation process and by treatment of preadipocytes with dexamethasone, conditions that were also associated with an increase in oleate transport. In contrast, the fibroblastic cell lines 3T3-C2 and L929, which do not differentiate, did not express the mRNAs at all stages of culture. The data suggest that FAT and CD36 belong to a family of proteins that bind/transport long-chain fatty acids or function as regulators of these processes.

Isolation and characteristics of a Bacillus subtilis mutant tolerant to the lytic action of sucrose esters of long-chain fatty acids

A spontaneous mutant of Bacillus subtilis 168, SR3, tolerant to the autolysis-inducing action of sucrose monoesters of long-chain fatty acids, was isolated. It was shown that its susceptibility to the lytic action of sucrose esters and glycerol esters of short-chain fatty acids, fatty acids themselves, some surfactants, uncouplers of oxidative phosphorylation, and β-lactam antibiotics against the mutant strain was similar to that of the wild-type strain. In the absence of sucrose monoesters, no substantial differences were observed between the mutant and the wild-type strains in cell autolysis and autolysin activity. It was found that in the mutant the cellular uptake of the molecules of sucrose ester of palmitic acid was suppressed. Also, a protein having a molecular weight of 41 kDa was richer in the membrane of strain SR3 than that of 168. The tolerance of the mutant to the lytic action of the ester is suggested to be derived from a decrease in the amount of ester molecules transferred into the membrane, where the activity of autolytic enzymes may be controlled.

Purification and Some Properties of Carboxylesterase fromArthrobacter globiformis; Stereoselective Hydrolysis of Ethyl Chrysanthemate

An esterase with excellent stereoselectivity for (+)-trans-ethyl chrysanthemate was purified to homogeneity from Arthrobacter globiformis SC-6-98-28. The purified enzyme hydrolyzed a mixture of ethyl chrysanthemate isomers stereoselectively to produce (+)-trans-acid with 100% stereoisomeric purity. The apparent molecular weight of the purified enzyme was 43,000 on SDS–polyacrylamide gel electrophoresis, and 94,000 on gel filtration chromatography. The optimum conditions for the ester hydrolysis were pH 10.0 at 45°C. The purified esterase hydrolyzed short-chain fatty acid esters, but did not have detectable activity on long-chain water-insoluble fatty acid esters. The enzyme activity was inbibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride.