Triester wax: a novel neutral lipid from the skin of the rhino mutant mouse.

A novel class of neutral lipids has been isolated from the skin of the rhino mutant mouse and has been characterized as a triester wax. The lipid, on saponification and transesterification, yielded fatty acids, omega-hydroxy fatty acids and 1,2-alkane diols. These products were identified by gas-liquid and thin-layer chromatography, infrared, nuclear magnetic resonance and mass spectroscopy, combined gas-liquid chromatography and mass spectrometry and chemical methods. Fatty acids were found to be predominantly of even chain length between C14 and C36 with highest concentration at C22 : 1. Hydroxy fatty acid methyl esters (trimethylsilyl ether derivatives) showed the presence of only three components in the relative abundance of 9: 70 : 21. The structure of the major component was established as 34-hydroxytetratricont-25-enoic acid and the other two components were characterized as 32-hydroxyditricont-23-enoic and 36-hydroxyhexatricont-27-enoic acids. In addition to these omega-9 unsaturates, other isomers having unsaturation at omega-7 and omega-8 were also present in small amounts. The 1,2-alkane diols were predominantly saturated in the range of C16-C24.     

2,3-Erythro-dihydroxyhexacosanoic acid and homologs: isolation from yeast cerebrin phosphate and determination of their structures

Homologs of methyl esters of very polar fatty acids were obtained by methanolysis of cerebrin phosphate isolated from baker's yeast. The major ester component was isolated by preparative gas-liquid chromatography and was found to be 2,3-dihydroxyhexacosanoic acid as deduced from the mass spectra of its trimethylsilyl ether and isopropylidene derivative, reaction with periodate, and comparison of its chromatographic behavior with that of synthetic erythro- and threo-dihydroxy acids. Its infrared spectrum supported the above conclusions. From their retention times by gas-liquid chromatography, the homologs were found to be saturated, unbranched 2,3-dihydroxy fatty acids with 24-27 carbon atoms. The synthesis of the new fatty acids, erythro- and threo-2,3-dihydroxyhexacosanoic acids, is also reported. A method for separating trans-2-hexacosenoic acid, a key intermediate of the above synthesis, and its isomer, trans-3-hexacosenoic acid, both formed by dehydrobromination of 2-bromohexacosanoic acid, is also described.     

Availability of Energy in Ester and Ether Derivatives of Glycols by Growing Chicks

Biological availability of 33 esters, 17 ethers and 2 acetals of ethanediol, 1,2-propanediol, 1,3-butanediol and 1,4-butanediol was compared by mini-test with chicks. Chicks can utilize esters of ethanediol, 1,2-propanediol and 1,3-butanediol with acetic acid and fatty acids of carbon chain length from 5 to 12 with more improved palatability than that of free acids, while availability of esters of these glycols with propionic and butyric acids was low. Esters of 1,4-butanediol and ether derivatives of these glycols was not available, except ethyl ether of di-ethanediol which was partially available. Acetacetal of ethanediol was partially available but n-butyracetal was not.     

Diaglycerol biosynthesis in everted sacs of rat intestinal mucosa.

The molecular specificity in the biosynthesis of diacylglycerols by rat intestinal mucosa was examined by means of radioactive markers, thin-layer chromatography with silver nitrate and gas-liquid chromatography with radioactivity monitoring. Bile salt micelles of alternately labeled monoacyglycerols and free fatty acids were incubated with everted sacs of intestinal mucosa for various periods of time and the diacylglycerols were isolated by solvent extraction and thin-layer chromatography. Sterospecific analyses of the X-1,2-diacylglycerols labeled from 2-monoacylglycerols showed that the sn-1,2-isomers (45-55%) were slightly in excess of the sn-2,3-isomers (34-45%) with the X-1,3-diacylglycerols accounting for the rest of the radioactivity (5-10%). This suggests that racemic diacylglycerols may be intermediates in the resynthesis of dietary fat in rat intestinal mucosa. Detailed analyses of the molecular species of the sn-1,2-diacylglycerols labeled from free fatty acids revealed that 10-45% of the total did not contain the acid present in the 2-monoacylglycerol supplied, and therefore had originated from the phosphatidic acid pathway. These findings are at variance with those obtained in isolated microsomes, which have suggested an inhibition of the phosphatidic acid pathway by monoacylglycerols as well as have given evidence of an exclusive synthesis of sn-1,2-diacylglycerols from 2-monoacylglycerols.     

Anaerobic n-Alkane Metabolism by a Sulfate-Reducing Bacterium, Desulfatibacillum aliphaticivorans Strain CV2803T

The alkane-degrading, sulfate-reducing bacterium Desulfatibacillum aliphaticivorans strain CV2803^T, recently isolated from marine sediments, was investigated for n-alkane metabolism. The total cellular fatty acids of this strain had predominantly odd numbers of carbon atoms (C odd) when the strain was grown on a C-odd alkane (pentadecane) and even numbers of carbon atoms (C even) when it was grown on a C-even alkane (hexadecane). Detailed analyses of those fatty acids by gas chromatography/mass spectrometry allowed us to identify saturated 2-, 4-, 6-, and 8-methyl- and monounsaturated 6-methyl-branched fatty acids, with chain lengths that specifically correlated with those of the alkane. Growth of D. aliphaticivorans on perdeuterated hexadecane demonstrated that those methyl-branched fatty acids were directly derived from the substrate. In addition, cultures on pentadecane and hexadecane produced (1-methyltetradecyl)succinate and (1-methylpentadecyl)succinate, respectively. These results indicate that D. aliphaticivorans strain CV2803^T oxidizes n-alkanes into fatty acids anaerobically, via the addition of fumarate at C-2. Based on our observations and on literature data, a pathway for anaerobic n-alkane metabolism by D. aliphaticivorans is proposed. This involves the transformation of the initial alkylsuccinate into a 4-methyl-branched fatty acid which, in addition to catabolic reactions, can alternatively undergo chain elongation and desaturation to form storage fatty acids.     

Biphenyl dioxolanes as circular dichroism probes for the assignment of absolute configuration to aliphatic diols: Extending the scope to anti 1,n-diols and cyclic syn 1,2-diols

We describe herein the use of a flexible biphenyl moiety as efficient chirality probe in the assignment of the absolute configuration (AC) of aliphatic, non-chromophoric diols. The diols are transformed in the corresponding biphenyl dioxolanes in which the biphenyl system has either a P or M torsion depending on the chirality of the diol. As the correlation between biphenyl torsion and diol AC has been established and the sense of torsion is revealed by the sign of the biphenyl A band at 250 nm in the CD spectrum of the dioxolane, then the diols AC can be assigned simply looking at the CD spectra of these derivatives. This approach proved to be general, straightforward, and reliable for anti 1,2- 1,3-, and 1,4-diols bearing both one and two stereogenic centers and for cyclic syn 1,2-diols.     

Relatively simple methodology for the determination of configuration of unsaturation of bacterial monounsaturated fatty acids: application to the unsaturates of Legionella spp.

Unsaturated fatty acids of known degree, position, and configuration of unsturation were esterified, and stereospecifically dihydroxylated at the double bond(s). cis-Hydroxylation was effected using Woodward's reagent (silver acetate/iodine/acetic acid), while trans-hydroxylation was effected using Fenton's reagent (hydrogen peroxide/ferrous sulfate/acetic acid). Diols derived from monounsaturated esters were recovered quantitatively, while tetraols derived from diunsaturates were lost, presumably during extensive washing. The stereospecifically dihydroxylated esters were analyzed by thin-layer chromatography on borate-impregnated silica gel plates, and by gas-liquid chromatography, on a nonpolar capillary column, as the trimethylsilyl, acetyl, n-butylboronyl, isopropylidene, and trifluoroacetyl derivatives. For each derivative, the erythro and threo diols were readily separable, and some resolution of positional isomers was observed. Thus, the cis/trans configuration, and in some instances, the position of unsaturation of the original monounsaturated fatty acid may be deduced. However, gas chromatography-mass spectrometry of appropriately derivatized diol esters is required for unambiguous determination of position of unsaturation in most cases. These reactions are simple, use readily available reagents, and require relatively little operator attention. Further, they do not require specialized apparatus, as do hydrogenation and ozonolysis, or potentially toxic chemicals, such as osmium tetroxide. This series of analyses was applied to the unsaturated non-hydroxylated fatty acids of Legionella species, which were shown to be monounsaturated and of the cis Δ9 family. Position of unsaturation was confirmed by gas chromatography-mass spectrometry.     

Microbial production of diols as platform chemicals: recent progresses

Diols are chemicals with two hydroxyl groups which have a wide range of appealing applications as chemicals and fuels. In particular, four diol compounds, namely 1,3-propanediol (1,3-PDO), 1,2-propanediol (1,2-PDO), 2,3-butanediol (2,3-BDO) and 1,4-butanediol (1,4-BDO) can be biotechnologically produced by direct microbial bioconversion of renewable materials. These diols are considered as platform green chemicals. We review and discuss here the recent development in the microbial production of these diols, especially regarding the engineering of production strains and optimization of the fermentation processes.     

Nonpolar lipids of a halotolerant species of Staphylococcus epidermidis.

The nonpolar lipids of a halotolerant Staphylococcus epidermidis, isolated in pure culture from a growth medium for extreme halophiles containing 25% sodium chloride, were found to contain squalene, menaquinone-7, free fatty acids (mainly anteiso-15:0 and anteiso-17:0), undecaprenol, nonaprenol with predominately cis-isoprene residues, heptaprenol, with predominately trans-isoprene residues, and 1,2- and 1,3-diglycerides containing anteiso- 15:0 and anteiso-17:0 branched chain fatty acid residues. The above compounds were isolated in pure form by column and thin-layer chromatography and were characterized by ultraviolet, proton magnetic resonance, and mass spectra. Fatty acid moieities were characterized by gas-liquid chromatographic retention times of their methyl esters.     

Degradation of Hydrocarbons by Members of the Genus Candida II. Oxidation of n-Alkanes and 1-Alkenes by Candida lipolytica

Candida lipolytica ATCC 8661 was grown in a mineral-salts hydrocarbon medium. n-Alkanes and 1-alkenes with 14 through 18 carbon atoms were used as substrates. Ether extracts of culture fluids and cells obtained from cultures grown on the various substrates were analyzed by thin-layer and gas-liquid chromatography. Analyses of fluids from cultures grown on n-alkanes indicated a predominance of fatty acids and alcohols of the same chain length as the substrate. In addition, numerous other fatty acids and alcohols were present. Analyses of saponifiable and nonsaponifiable material obtained from the cells revealed essentially the same products. The presence of primary and secondary alcohols, as well as fatty acids, of the same chain length as the n-alkane substrate suggested that attack on both the methyl and α-methylene group was occurring. The significance of these two mechanisms in the degradation of n-alkanes by this organism was not evident from the data presented. Analyses of fluids from cultures grown on 1-alkenes indicated the presence of 1,2-diols, as well as ω-unsaturated fatty acids, of the same chain length as the substrate. Alcohols present were all unsaturated. Saponifiable and nonsaponifiable material obtained from cells contained essentially the same products. The presence of 1,2-diols and ω-unsaturated fatty acids of the same chain length as the substrate from cultures grown on 1-alkenes indicated that both the terminal methyl group and the terminal double bond were being attacked.     

High-performance liquid chromatography of fatty acid isopropylidene hydrazides and its application in lipid analysis

Fatty acid isopropylidene hydrazides, prepared by stepwise treatment of acyl lipids with hydrazine and acetone, were analyzed by high-performance liquid chromatography on a reversed-phase column. These derivatives could be easily eluted with 15% water in methanol and monitored by measuring absorbance at 229 nm with a uv detector. Their elution behavior, in general, was similar to that of methyl esters and some commonly used ultraviolet-absorbing derivatives of fatty acids. The new method has been used for fatty acid analysis of some oils.     

Quantitative analysis of retinal glycerolipid molecular species acetylated by acetolysis

A method for the quantitative analysis of molecular species of 1,2-diacylglycerol acetates (1,2-DGAC) containing polyunsaturated fatty acids is described. Phosphatidylethanolamine (PE) isolated from frog retina was used to test the method. PE was converted to 1,2-DGAC by acetolysis. The molecular species of the 1,2-DGAC were resolved by reverse-phase high performance liquid chromatography (HPLC), detected by UV absorption spectroscopy at 210 nm, and identified by gas-liquid chromatography (GLC) of the fatty acid methyl esters (FAME). Molar response curves were generated for each DGAC molecular species that eluted as a single entity from HPLC by determining the moles of fatty acids in the molecular species collected and the response (peak area unit) of the UV detector. Each molecular species response curve was linear from about 10 pmoles to 4-8 nmoles, allowing the slope of each curve to be used as a molar absorptivity. This method provides a means for quantification of most of the molecular species of all glycerolipid classes.     

A gas-liquid chromatographic method for steric analysis of epoxy acids

A gas-liquid chromatographic method for determination of the absolute configuration of the two chiral carbon atoms of epoxy fatty acids was developed. The method involved (1) conversion of the saturated epoxy ester into a pair of regioisomeric allylic alcohols by consecutive treatments with selenophenoxide anion and hydrogen peroxide, (2) oxidative ozonolysis performed on the (-)-menthoxycarbonyl derivatives of the allylic alcohols, and (3) steric analysis of the resulting two 2-hydroxy acids (methyl esters, (-)-menthoxycarbonyl derivatives) by gas-liquid chromatography using appropriate reference compounds. Application of the method for steric analysis of several synthetic epoxyoctadecanoates as well as (+)-vernolic acid derived from Vernonia galamensis is described.     

Determination of trace levels of fatty acid metal salts by high-performance liquid chromatography with fluorescence prelabeling

A simple method is described for picomole determinations of fatty acid metal salts. Fatty acid salts are directly labeled with 4-bromomethyl-7-methoxycoumarin in the presence of excess ethylenediaminetetraacetic acid tripotassium salt without any solvent extractions. The fluorescence derivatives of fatty acids are separated by reverse-phase high-performance liquid chromatography followed by fluorometric detection. The response of each fatty acid (C8-C18) calcium salt is linear from 1 to 50 micrograms/ml of samples. The detection limit is about 7 pmol. Good recoveries are obtained for the calcium salts of myrystic acid and soap (C8-C18, C18:1,2). The new method is successfully applied to the study on biodegradation of fatty acids in river water.     

Biosynthesis of diacylglycerols by rat intestinal mucosa in vivo.

The biosynthesis of diacylglycerols was studied in rat intestinal mucosa during in vivo absorption of a low molecular weight fraction fraction of butter oil and of the corresponding medium and long chain fatty acids. The experimental fat solutions were given by stomach tube to the animals after a 24-h fast and mucosal scraping were collected 3 h later. The lipids were isolated and the acylclycerols determined by combined thin-layer chromatography gas-liquid chromatography techniques and stereospecific analyses. Free fatty acid feeding led mainly to sn-1,2-diacyl-glycerols, which contained exogenous and endogenous fatty acids. During triacylglycerol feeding, both sn-1,2-and sn-2,3-diacylglycerols were recovered in significant amounts from the intestinal mucosa. The composition of the sn-2,3-diacylglycerols corresponded to that with exogenous fatty acids but the sn-1,2-diacylglycerols clearly contained both exogenous and endogenous fatty acids. In all cases it was possible to isolate endogenous sn-1,2-diacylglycerols made up largely of species with linoleic and arachidonic acids in the 2 position and palmitic and stearic acids in the 1 position, which apparently were not converted to triacylglycerols. The in vivo reacylation of 2-monoacylglycerols via both sn-1,2- and sn-2,3-diacylglycerols is in agreement with similar findings in vitro with everted sacs of rat intestinal mucosa.     

Acyl specificity in triglyceride synthesis by lactating rat mammary gland.

We have studied the specificity of the acyl-CoA:diglyceride acyltransferase reaction in lactating rat mammary gland to provide a rational explanation at the enzyme level for the nonrandom distribution of fatty acids in milk fat triglycerides. Acyl-CoA:diglyceride acyltransferase activity was measured using various diglyceride and radioactive acyl-CoA substrates; products were identified as triglycerides by thin-layer and gas-liquid chromatography. Most of the enzymatic activity was located in the microsomal fraction and showed a broad specificity for the acyl donors tested C10, C12, C14, C16, C18, and C18:1 CoA esters). The acyltransferase activity was highly specific for sn-1,2-diglyceride enantiomers; rac-1,3- and sn-2,3-diglycerides were relatively inactive. The acyl-CoA specificity was not affected by the type of 1,2-diglyceride acceptor offered, although dilaurin was the best acceptor and sn-1,2-dilaurin greater than sn-1,2-dimyristin greater than sn-1,2-dipalmitin greater than sn-1,2-distearin. We have previously shown that in the microsomal fraction from lactating rat mammary gland, the acyltransferase activities concerned with the conversion of sn-glycero-3-phosphate to diacylglycerophosphate show a very marked specificity for long chain acyl-CoA's. Therefore, we conclude that the predominant localization of long chain fatty acids in the 1 and 2 positions, and of shorter chain fatty acids in the 3 position of the glycerol backbone, results at least in part from the specificities of the mammary gland acyltransferases.     

Amines in unicellular green algae: 2. Amines in Scenedesmus acutus

Scenedesmus acutus contains about 10 major amines and at least 20 other amines which are present in very small quantities. The following amines were identified by mass spectrometry after separation of the trifluoroacetyl derivatives by gas-liquid chromatography and of the dansyl ² derivatives by thin-layer chromatography: methylamine, dimethylamine, ethylamine, ethanolamine, putrescine, cadaverine, spermidine, N-(3-aminopropyl)-1,3-diaminopropane, N-(4-aminobutyl)-1,4-diaminobutane, 2-phenylethylamine, tyramine, piperazine, adenine, and γ-butyrolactam. The methods applied for the analyses of these amines are described and discussed.     

Determination of the molecular species composition of diacylglycerols in human adipose tissue by solid-phase extraction and gas chromatography on a polar phase

The free diacylglycerols (DAGs) in adipose tissue are involved in the metabolism of stored lipids and hence are related to the supply of fatty acids for other tissues. This paper describes a simple, fast, and reproducible method for the identification and quantification of different molecular species of DAGs in human adipose tissue. The method comprised solid-phase extraction on a diol-bonded phase column combined with capillary GC analysis of silylated DAG derivatives on a polar phase (65% phenylmethylsilicone). Separation of the DAGs was achieved based on chain length, isomeric structure (1,2- and 1,3-DAGs), and degree of unsaturation. The main DAGs were 1,2-OO, 1,2-OP, 1,2-LO and 1,2-LP. The composition was corroborated by analysis of the component fatty acids of the DAGs, 18:1(n-9), 16:0, and 18:2(n-6) being the three major fatty acids obtained.     

Alcohols produce reversible and irreversible acceleration of phospholipid flip-flop in the human erythrocyte membrane.

The slow, non-mediated transmembrane movement of the lipid probes lysophosphatidylcholine, NBD-phosphatidylcholine and NBD-phosphatidylserine in human erythrocytes becomes highly enhanced in the presence of 1-alkanols (C2-C8) and 1,2-alkane diols (C4-C8). Above a threshold concentration characteristic for each alcohol, flip rates increase exponentially with the alcohol concentration. The equieffective concentrations of the alcohols decrease about 3-fold per methylene added. All 1-alkanols studied are equieffective at comparable calculated membrane concentrations. This is also observed or the 1,2-alkane diols, albeit at a 5-fold lower membrane concentration. At low alcohol concentrations, flip enhancement is reversible to a major extent upon removal of the alcohol. In contrast, a residual irreversible flip acceleration is observed following removal of the alcohol after a treatment at higher concentrations. The threshold concentrations to produce irreversible flip acceleration by 1-alkanols and 1,2-alkane diols are 1.5- and 3-fold higher than those for flip acceleration in the presence of the corresponding alcohols. A causal role in reversible flip-acceleration of a global increase of membrane fluidity or membrane polarity seems to be unlikely. Alcohols may act by increasing the probability of formation of transient structural defects in the hydrophobic barrier that already occur in the native membrane. Membrane defects responsible for irreversible flip-acceleration may result from alterations of membrane skeletal proteins by alcohols.     

Anaerobic transformation of alkanes to fatty acids by a sulfate-reducing bacterium,strain Hxd3

Strain Hxd3, an alkane-degrading sulfate reducer previously isolated and described by Aeckersberg et al. (F. Aeckersberg, F. Bak, and F. Widdel, Arch. Microbiol. 156:5-14, 1991), was studied for its alkane degradation mechanism by using deuterium and (13)C-labeled compounds. Deuterated fatty acids with even numbers of C atoms (C-even) and (13)C-labeled fatty acids with odd numbers of C atoms (C-odd) were recovered from cultures of Hxd3 grown on perdeuterated pentadecane and [1,2-(13)C(2)]hexadecane, respectively, underscoring evidence that C-odd alkanes are transformed to C-even fatty acids and vice versa. When Hxd3 was grown on unlabeled hexadecane in the presence of [(13)C]bicarbonate, the resulting 15:0 fatty acid, which was one carbon shorter than the alkane, incorporated a (13)C label to form its carboxyl group. The same results were observed when tetradecane, pentadecane, and perdeuterated pentadecane were used as the substrates. These observations indicate that the initial attack of alkanes includes both carboxylation with inorganic bicarbonate and the removal of two carbon atoms from the alkane chain terminus, resulting in a fatty acid one carbon shorter than the original alkane. The removal of two terminal carbon atoms is further evidenced by the observation that the [1,2-(13)C(2)]hexadecane-derived fatty acids contained either two (13)C labels located exclusively at their acyl chain termini or none at all. Furthermore, when perdeuterated pentadecane was used as the substrate, the 14:0 and 16:0 fatty acids formed both carried the same numbers of deuterium labels, while the latter was not deuterated at its carboxyl end. These observations provide further evidence that the 14:0 fatty acid was initially formed from perdeuterated pentadecane, while the 16:0 fatty acid was produced after chain elongation of the former fatty acid with nondeuterated carbon atoms. We propose that strain Hxd3 anaerobically transforms an alkane to a fatty acid through a mechanism which includes subterminal carboxylation at the C-3 position of the alkane and elimination of the two adjacent terminal carbon atoms.