Skin surface lipids of the mole Scalopus aquaticus

Skin surface lipids of the mole Scalopus aquaticus were found to consist principally of squalene (70%), wax esters (15%), and sterol esters (5%), together with small amounts of triglycerides, free fatty acids, free fatty alcohols, and free sterols. Analysis of the fatty acids occurring free and as wax esters and sterol esters showed these to consist of approximately equal amounts of saturated and monounsaturated compounds. The saturated fatty acids consisted predominantly of odd-carbon anteiso and even-carbon straight-chain compounds, with minor amounts of even-carbon iso-branched chains. The unsaturated fatty acids had double bond positions that would have been produced by delta 9-desaturation of C14, C16 and C18 straight chain saturated precursors. Both the free and the esterified fatty alcohols had chain structures corresponding with those of the fatty acids but of somewhat greater average chain length. Discovery of a major proportion of squalene in the sebum of this animal extends the number of non-human species that have this characteristic to four, all of which inhabit a damp environment, suggesting that squalene conveys some biological advantage under these conditions.     

Biosynthesis of wax esters in tissues of Sinapis alba L. seeds

The biosynthesis of wax esters has been investigated in maturing seeds of Sinapis alba. Exogenous long-chain alcohols are incorporated exclusively into alkyl moieties of wax esters. Oxidation of the long-chain alcohols is not detected. Exogenous fatty acids are incorporated into acyl moieties of wax esters to a low extent. A reduction of fatty acids to alcohols is not observed. Synthesis of wax esters is localized exclusively in the testa; both outer and inner integument are equally active in wax ester biosynthesis. The biosynthesis of wax esters is specific with regard to both chain length and degree of unsaturation of long-chain alcohols. Exogenous and endogenous sterols are not esterified.     

Quantitative analysis of fatty acid precursors in marine samples: direct conversion of wax ester alcohols and dimethylacetals to FAMEs

To apply fatty acid analyses to the study of foraging ecology and diet determination, all compounds that may be deposited as fatty acids in a predator must be quantified in the prey. These compounds include the usual fatty acids in acyl lipids, but also the alcohols of wax esters and the vinyl ethers of plasmalogens. In routine fatty acid analysis, samples are extracted and transesterified (methylated), resulting in the formation of fatty acid methyl esters (FAMEs); however, fatty alcohols and dimethylacetals (DMAs) are also generated if wax esters or plasmalogens are present. Here, we present a new method using a modified Jones' reagent to oxidize these alcohols and DMAs to free fatty acids (FFAs). These FFAs are then easily methylated and quantitatively recombined with FAMEs from the same sample. This generates a fatty acid signature of prey that is equivalent to that which the predator has available for deposition upon digestion of that prey. This method is validated with alcohol and DMA standards. Its application to typical marine samples is also presented, demonstrating the change in effective fatty acid signature after inclusion of fatty acids derived from wax esters and plasmalogens.     

Properties of alkyl hydroxycinnamates and effects on Pseudomonas fluorescens.

Alkyl esters of six hydroxycinnamic acids, shown to be active antimicrobial agents when tested against Pseudomonas fluorescens, were further investigated for their effects against this organism. There was no statistically significant adaptation by this organism to either of the methyl esters of caffeic, rho-coumaric, cinnamic, or rho-hydroxybenzoic acids. Mixtures of these compounds taken two at a time gave at least additive effects, with some mixtures showing synergism. Preliminary work was also performed to determine the mode of inhibitory action for these compounds. The inhibition of oxygen utilization by the methyl esters correlated well with growth inhibition. Short-term lethality studies showed that none of the alkyl esters or methyl or propyl paraben produced any bacteriocidal effects. Oil-water partition coefficients were determined for these compounds and were shown to have no correlation with growth inhibitions. These all point to a specific mode of action, based in part on cellular energy depletion, rather than the nonspecific membrane-disrupting effects of other phenolic antimicrobial agents.     

Nutritional significance and metabolism of very long chain fatty alcohols and acids from dietary waxes

Very long chain fatty alcohols obtained from plant waxes and beeswax have been reported to lower plasma cholesterol in humans. This review discusses nutritional or regulatory effects produced by wax esters or aliphatic acids and alcohols found in unrefined cereal grains, beeswax, and many plant-derived foods. Reports suggest that 5-20 mg per day of mixed C24-C34 alcohols, including octacosanol and triacontanol, lower low-density lipoprotein (LDL) cholesterol by 21%-29% and raise high-density lipoprotein cholesterol by 8%-15%. Wax esters are hydrolyzed by a bile salt-dependent pancreatic carboxyl esterase, releasing long chain alcohols and fatty acids that are absorbed in the gastrointestinal tract. Studies of fatty alcohol metabolism in fibroblasts suggest that very long chain fatty alcohols, fatty aldehydes, and fatty acids are reversibly inter-converted in a fatty alcohol cycle. The metabolism of these compounds is impaired in several inherited human peroxisomal disorders, including adrenoleukodystrophy and Sj?gren-Larsson syndrome. Reports on dietary management of these diseases confirm that very long chain fatty acids (VLCFA) are normal constituents of the human diet and are synthesized endogenously. Concentrations of VLCFA in blood plasma increase during fasting and when children are placed on ketogenic diets to suppress seizures. Existing data support the hypothesis that VLCFA exert regulatory roles in cholesterol metabolism in the peroxisome and also alter LDL uptake and metabolism.     

Lumenal hydrolysis of menhaden and rapeseed oils and their fatty acid methyl and ethyl esters in the rat

Simple alkyl (ethyl) esters of polyunsaturated fish oil fatty acids have been proposed as dietary supplements, but their relative efficiency of digestion and absorption have not been determined. Using stomach tubes, we gave rats menhaden or rapeseed oils, or the corresponding methyl and ethyl esters, and determined by chromatographic methods the lipid classes and molecular species recovered from the lumen of the jejunum during the first 1 to 2.5 h of digestion. Hydrolysis of menhaden oil resulted in a preferential retention of a high proportion of the polyunsaturated long chain acids in the sn-2-monoacylglycerols and in the residual triacyglycerols, while digestion of rapeseed oil led to a preferential release of free long chain monounsaturated fatty acids. In contrast, hydrolysis of the alkyl (methyl and ethyl) esters of the fatty acids of either menhaden or rapeseed oil resulted in a composition of free fatty acids which was much more representative of the original esters. It was therefore concluded that the differential lumenal liberation of the long chain and polyunsaturated (three or more double bonds) fatty acids from fish and rapeseed oil is largely due to their characteristic distribution between the primary and secondary positions in the glycerol molecule, and to a much lesser extent to a chain length discrimination by pancreatic lipase. This study also shows that the methyl and ethyl esters are hydrolyzed about 4 times more slowly than the corresponding triacylglycerols, which is sufficient to maintain a saturated micellar solution of fatty acids in the intestinal lumen during absorption.     

Malonyl-CoA and long chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion.

Several approaches were used to test the hypothesis proposing a role for acyl-CoA esters in nutrient-induced insulin release (Prentki, M., and Matschinsky, F. M. (1987) Physiol. Rev. 67, 1185-1248; Corkey, B. E., Glennon, M. C., Chen, K. S., Deeney, J. T., Matschinsky, F. M., and Prentki, M. (1989) J. Biol. Chem. 264, 21608-21612). Exogenous saturated long chain fatty acids markedly potentiated glucose-induced insulin release and elevated long chain acyl-CoA esters in the clonal beta-cell line (HIT). The secretory action depended on the fatty acid chain length, occurred in the range 3-20 microM (free concentration of palmitate), and was reversible and inhibitable by the neuromodulator somatostatin. 2-Bromopalmitate, an inhibitor of carnitine palmitoyl transferase I, suppressed the oxidation of endogenous fatty acids and promoted release of insulin. Only the nutrients or the combination of nutrients that caused secretion elevated malonyl-CoA. The short-chain acyl-CoA profile of HIT cells stimulated by various nutrients was determined in the presence of the nonstimulatory fuel glutamine. Glucose and leucine each provoked similar changes in acyl-CoA compounds. Both secretagogues elevated malonyl-CoA 3-6-fold, whereas succinyl-CoA, free CoASH, acetyl-CoA, and the free CoASH to acetyl-CoA ratio remained unaltered. Furthermore, only when inhibition of fatty acid oxidation was associated with a rise in malonyl-CoA did the total (mitochondrial plus cytoplasmic) content of long chain acyl-CoA esters correlate inversely with insulin release promoted by various nutrients. The results are consistent with the concept that fuel stimuli cause a rise in malonyl-CoA which by inhibiting fatty acid oxidation increase cytosolic long chain acyl-CoA esters. These data provide further support for a model in which malonyl-CoA and long chain acyl-CoAs esters serve as metabolic coupling factors when pancreatic beta-cells are stimulated with glucose and other nutrient secretagogues.     

Synthesis of wax esters by a cell-free system from barley (Hordeum vulgare L.)

Experimental evidence for a membranebound microsomal ester synthetase from Bonus barley primary leaves is reported. The results are consistent with at least two mechanisms for the synthesis of barley wax esters: an acyl-CoA-fattyalcohol-transacylase-type reaction and an apparent direct esterification of alcohols with fatty acids. Biosynthesis of wax esters was not specific with regard to the chain length of the tested alcohols. The microsomal preparation readily catalyzed the esterification of C₁₆-, C₁₈-, C₂₂- or C₂₄-labelled alcohols with fatty acids of endogenous origin. Exogenous long-chain alcohols were exclusively incorporated into the alkyl moieties of the esters. Addition of ATP, CoA and-or free fatty acids was not effective in stimulating or depressing the esterifying activity of the microsomal fraction. Partial solubilization of the ester synthetase was obtained using phosphate-buffered saline.Abbreviations P pellet - PBS phosphate-buffered saline - S supernatant - SDS sodium dodecyl sulphate     

Lipids as renewable resources: current state of chemical and biotechnological conversion and diversification

Oils and fats are the most important renewable raw materials of the chemical industry. They make available fatty acids in such purity that they may be used for chemical conversions and for the synthesis of chemically pure compounds. Oleic acid (1) from “new sunflower,” linoleic acid (2) from soybean, linolenic acid (3) from linseed, erucic acid (4) from rape seed, and ricinoleic acid (5) from castor oil are most important for chemical transformations offering in addition to the carboxy group one or more C-C-double bonds. New plant oils containing fatty acids with new and interesting functionalities such as petroselinic acid (6) from Coriandrum sativum, calendic acid (7) from Calendula officinalis, α-eleostearic acid (8) from tung oil, santalbic acid (9) from Santalum album (Linn.), and vernolic acid (10) from Vernonia galamensis are becoming industrially available. The basic oleochemicals are free fatty acids, methyl esters, fatty alcohols, and fatty amines as well as glycerol as a by-product. Their interesting new industrial applications are the usage as environmentally friendly industrial fluids and lubricants, insulating fluid for electric utilities such as transformers and additive to asphalt. Modern methods of synthetic organic chemistry including enzymatic and microbial transformations were applied extensively to fatty compounds for the selective functionalization of the alkyl chain. Syntheses of long-chain diacids, ω-hydroxy fatty acids, and ω-unsaturated fatty acids as base chemicals derived from vegetable oils were developed. Interesting applications were opened by the epoxidation of C-C-double bonds giving the possibility of photochemically initiated cationic curing and access to polyetherpolyols. Enantiomerically pure fatty acids as part of the chiral pool of nature can be used for the synthesis of nonracemic building blocks.     

Epicuticular wax composition in relation to aphid infestation and resistance in red raspberry (Rubus idaeus L.)

Epicuticular waxes from the aphid-resistant red raspberry (Rubus idaeus) cultivar Autumn Bliss and the aphid-susceptible cultivar Malling Jewel were collected from the newly emerging crown leaves, and also from the group of four more mature leaves immediately below the crown. Resistance and susceptibility status of the leaves to infestation by the large raspberry aphid, Amphorophora idaei, were determined by bioassay with the insect just prior to collection of the wax. Analysis showed the waxes to consist of a complex mixture of free fatty acids; free primary alcohols and their acetates; secondary alcohols; ketones; terpenoids including squalene, phytosterols, tocopherol and amyrins; alkanes and long chain alkyl and terpenyl esters. Compositional differences which may relate to A. idaei-resistance status were noticeably higher levels of sterols, particularly cycloartenol, together with the presence of branched alkanes, and an absence of C₂₉ ketones and the symmetrical C₂₉ secondary alcohol in wax from the resistant cultivar Bliss. There were also differences between the cultivars in the distribution of individual amyrins and tocopherols and in the chain length distribution for homologues of fatty acids, primary alcohols and alkanes, and these may also be related to resistance to A. idaei. Emerging leaves had lower levels of primary alcohols and terpenes, but higher levels of long-chain alkyl esters, and in general, more compounds of shorter chain-length than the more mature leaves. During bioassay A. idaei displayed a preference to settle on the more mature leaves. This may be due to greater wax coverage and higher levels of the compounds of shorter chain length found in the newly emerged younger leaves at the crown of the plant.     

CHEMICAL STRUCTURE AND NARCOTIC POTENCY TO GRAIN WEEVILS. ALCOHOLS AND METHYL ESTERS OF FATTY ACIDS

The narcotic and toxic potencies of the first seven homologous alcohols and the methyl esters of the first seven homologous fatty acids have been determined using grain weevils (Calandra granaria). The experiments were designed as balanced incomplete blocks, and Finney's probit plane technique was applied to the results. In both series of compounds the narcotic and toxic potencies expressed in thermodynamic concentrations decreased with increasing number of carbon atoms. The margin between narcotic dose and toxic dose is greater in the alcohol series than in the ester series. The methyl esters of the fatty acids showed a stepwise descent in biological potency, the odd numbered series being more active than the even series. The penetration of these compounds into grain weevils and their biological action are discussed in relation to the coefficients of response to log concentration and log time of exposure.     

Picolinyl esters for the structural determination of fatty acids by GC/MS

The position of unsaturation, chain branching, and other structural features of fatty acids are not often apparent from the mass spectra of common derivatives such as methyl esters because of factors such as charge location at the carboxy termiunus and migration of double bonds. The spectra of picolinyl esters, on the other hand, contain fragment ions that provide this information. The esters are synthesized by reaction of the acids with thionyl chloride to form the acid chloride that is reacted with 3-pyridylcarbinol to give the ester. Under electron impact conditions in the mass spectrometer, an electron is removed from the nitrogen of the pyridine ring and a hydrogen atom is abstracted from the alkyl chain to this electron-deficient site. This process produces a radical site in the chain that initiates chain cleavage. Hydrogen atoms can be removed from any position of the chain with varying probability, depending on the chain structure. Thus, diagnostic ions are produced from each type of fatty acid whose masses and relative abundances reflect the structure of the alkyl chain and any substituents. Patterns of fragmentation for straight-chain, branched-chain, unsaturated and cyclic fatty acids are described together with those containing hydroxy-, epoxy-, keto-, and ether groups.     

Lipolysis of menhaden oil triacylglycerols and the corresponding fatty acid alkyl esters by pancreatic lipase in vitro: a reexamination

In order to distinguish between possible fatty acid differences during lumenal lipolysis and cellular absorption, we have reinvestigated the in vitro hydrolysis of menhaden oil and its alkyl esters by pancreatic lipase. For this purpose we incubated menhaden oil or its fatty acid methyl and ethyl esters with porcine pancreatic lipase in the presence of bile salts and determined the composition of the released free fatty acids, monoacylglycerols, diacylglycerols, and residual triacylglycerols, or the free fatty acids and residual alkyl esters, respectively, by thin-layer and gas-liquid chromatography. There was significant discrimination against the delta 4- to delta 7-unsaturated fatty acids of both medium and long chain lengths during the hydrolysis of menhaden oil and its fatty acid ethyl esters. In general, the ethyl esters were hydrolyzed 10-50 times more slowly than the corresponding glyceryl esters, depending on the exact ratio of the two substrate types. None of the triacylglycerols or ethyl esters, however, was completely resistant to hydrolysis resulting in an eventual cleavage of all the alkyl esters and presumably all the primary ester bonds in the triacylglycerol molecules. Since the rate of release of the least resistant fatty acid exceeded that of the most resistant acid by only a factor of 6, it is concluded that in the presence of a large excess of lipase the liberated fatty acids would approach the composition of the dietary alkyl or glyceryl esters, as observed during lumenal lipolysis (Yang, L.-Y., A. Kuksis, and J. J. Myher. 1989. Biochem. Cell Biol. 67: 192-204).     

Identification by gas chromatography and mass spectrometry of lipids from the rat Harderian gland

Lipids from rat Harderian glands were extracted with ethyl acetate, hydrolysed with base and examined by gas chromatography (GC) and gas chromatography—mass spectrometry (GC—MS) as trimethylsilyl (TMS), [²H₉]TMS, methyl ester—TMS, picolinyl, nicotinate and nicotinylidene derivatives. The latter three derivatives were used to reveal the structures of the alkyl chains of fatty acids, alcohols and glycerol ethers, respectively. Forty-eight compounds were identified, representing about 97% of the total extracted lipids as measured by GC peak areas. The major constituents were fatty acids with chain lengths from 12 to 22 carbon atoms (mainly C₁₈ and C₂₀) and fatty alcohols (C₁₆ to C₂₆) derived from wax esters. Most of these acids and alcohols were unsaturated in the ω-7 position and were accompanied by smaller amounts of the saturated and ω-5 monounsaturated analogues. Glycerol ethers were also identified for the first time in this secretion; the ether chains contained from 14 to 19 carbon atoms (mainly 16) and were straight-chain saturated, unsaturated (ω-5 and ω-7) and branched (iso). The only sterol found was cholesterol amounting to 1.24% of the total extract.     

Preparation of C60-based active esters and coupling of C60 moiety to amines or alcohols

We report the syntheses of C(60)-based active esters and the coupling of their C(60) moiety to various amines or alcohols. Methano[60]fullerene carboxylic acid was activated by esterification with N-hydroxysuccinimide (NHS) or pentafluorophenol (PFP) and the active esters were isolated. Reactions of the active esters with amines or alcohols proceeded easily to give a variety of compounds having the C(60) moiety.     

Regioselective oxygenation of fatty acids, fatty alcohols and other aliphatic compounds by a basidiomycete heme-thiolate peroxidase

Reaction of fatty acids, fatty alcohols, alkanes, sterols, sterol esters and triglycerides with the so-called aromatic peroxygenase from Agrocybe aegerita was investigated using GC-MS. Regioselective hydroxylation of C(12)-C(20) saturated/unsaturated fatty acids was observed at the ω-1 and ω-2 positions (except myristoleic acid only forming the ω-2 derivative). Minor hydroxylation at ω and ω-3 to ω-5 positions was also observed. Further oxidized products were detected, including keto, dihydroxylated, keto-hydroxy and dicarboxylic fatty acids. Fatty alcohols also yielded hydroxy or keto derivatives of the corresponding fatty acid. Finally, alkanes gave, in addition to alcohols at positions 2 or 3, dihydroxylated derivatives at both sides of the molecule; and sterols showed side-chain hydroxylation. No derivatives were found for fatty acids esterified with sterols or forming triglycerides, but methyl esters were ω-1 or ω-2 hydroxylated. Reactions using H(2)(18)O(2) established that peroxide is the source of the oxygen introduced in aliphatic hydroxylations. These studies also indicated that oxidation of alcohols to carbonyl and carboxyl groups is produced by successive hydroxylations combined with one dehydration step. We conclude that the A. aegerita peroxygenase not only oxidizes aromatic compounds but also catalyzes the stepwise oxidation of aliphatic compounds by hydrogen peroxide, with different hydroxylated intermediates.     

The PMR analysis of non-conjugated alkenoic and alkynoic acids and esters.

The 220 MHz PMR spectra of 143 non-conjugated alkenoic and alkynoic acids and esters are correlated so as to provide a method for the structural analysis of such compounds in general. The spectral data are explained in terms of long-range deshielding of the double bonds, triple bonds, acid and ester groups in the molecules, and parameters are derived to quantify the influence of these groups on the chemical shifts of methyl and methylene protons up to six carbon atoms distant along an alkyl chain. It is shown that, by the application of these parameters, 220 MHz PMR spectroscopy can be used to determine both the stereochemistry and position of double bonds, and the position of triple bonds, in the majority of fatty acids and esters. The 2- to 9- and 13- to 17-cis- and trans-isomers of octadecenoic acid may be readily idenfited in this way, whilst for the octadecynoic acids all positional isomers may be characterized. Examples are also given of the structural analysis of several polyenoic compounds, including methyl cis-5, cis-8, cis-11, cis-14, cis-5, cis-8, cis-11, trans-14, and trans-5, cis-8, cis-11, cis-14-eicosatetraenoates, and methyl trans-5, cis-9, cis-12-octadecatrienoate.     

Nanotubules on plant surfaces: chemical composition of epicuticular wax crystals on needles of Taxus baccata L

Needles of Taxus baccata L. were covered with tubular epicuticular wax crystals varying in diameters (100 and 250 nm) and lengths (300-500 and 500-1000 nm) on the abaxial and adaxial surfaces, respectively. Various sampling protocols were employed to study the chemical composition of the needle waxes on three different levels of spatial resolution. First, a dipping extraction of whole needles yielded the total cuticular wax mixture consisting of very long chain fatty acids (21%), alkanediols (19%), phenyl esters (15%), and secondary alcohols (9%) together with small amounts of aldehydes, primary alcohols, alkanes, alkyl esters, and tocopherols. Second, waxes from both sides of the needle were sampled separately by brushing with CHCl3-soaked fabric glass. Both sides showed very similar qualitative composition, but differed drastically in quantitative aspects, with nonacosan-10-ol (18%) and alkanediols (33%) dominating the abaxial and adaxial waxes, respectively. Third, the epi- and intracuticular wax layers were selectively sampled by a combination of mechanical wax removal and brushing extraction. This provided direct evidence that the tubular wax crystals contained high percentages of nonacosane-4,10-diol and nonacosane-5,10-diol on the abaxial surface, and nonacosan-10-ol on the adaxial surface of the needles. Together with these compounds, relatively large amounts of fatty acids and smaller percentages of aldehydes, primary alcohols, alkyl esters, and alkanes co-crystallized in the epicuticular layer. In comparison, the intracuticular wax consisted of higher portions of cyclic constituents and aliphatics with relatively high polarity. The formation of the tubular crystals is discussed as a spontaneous physico-chemical process, involving the establishment of gradients between the epi- and intracuticular wax layers and local phase separation.     

Monolayer and calorimetric studies of phosphatidylcholines containing branched-chain fatty acids and of their interactions with cholesterol and with a bacterial hopanoid in model membranes

Although methyl iso- and anteiso-branched fatty acids occur widely in the membrane lipids of prokaryotic microorganisms, relatively little is known about the physical properties of phospholipids containing these fatty acids. We report here a monolayer and differential scanning calorimetric characterization of several synthetic phosphatidylcholines containing branched-chain fatty acids, and describe the interactions of these phospholipids with cholesterol and with a bacterial hopanoid. We find that monolayers as well as bilayers of methyl isobranched- and especially of methyl anteisobranched-fatty-acid-containing phosphatidylcholines exhibit a reduced solid-to-fluid phase transition temperature in comparison with linear saturated fatty acid-containing phosphatidylcholines of comparable chain length. We also find that the liquid-condensed or gel states of branched-chain fatty acid-containing phosphatidylcholines are partially disordered relative to those of phospholipids containing linear saturated fatty acids, although the presence of a methyl branch has only a small effect on hydrocarbon chain packing in the liquid-expanded or liquid-crystalline states. The presence of cholesterol was found to produce a marked condensation of liquid-expanded films and a small condensation of liquid-condensed films, whether the phosphatidylcholine contained linear or branched-chain fatty acyl constituents. The presence of a bacterial hopanoid produced similar, although slightly smaller, monolayer-condensing effects, indicating that these compounds may perform a cholesterol-like function in bacterial membranes.