Antifungal 3-hydroxy fatty acids from Lactobacillus plantarum MiLAB 14

We report the identification and chemical characterization of four antifungal substances, 3-(R)-hydroxydecanoic acid, 3-hydroxy-5-cis-dodecenoic acid, 3-(R)-hydroxydodecanoic acid and 3-(R)-hydroxytetradecanoic acid, from Lactobacillus plantarum MiLAB 14. The concentrations of the 3-hydroxy fatty acids in the supernatant followed the bacterial growth. Racemic mixtures of the saturated 3-hydroxy fatty acids showed antifungal activity against different molds and yeasts with MICs between 10 and 100 micrograms ml-1.     

Nature and location of amide-bound (R)-3-acyloxyacyl groups in lipid A of lipopolysaccharides from various gram-negative bacteria

It has previously been demonstrated [Eur. J. Biochem. 124, 191-198 (1982) and 137, 15-22 (1983)] that the lipid A component of Salmonella and Proteus lipopolysaccharides contains amide-linked (R)-3-acyloxyacyl residues. In the present study lipid A of other gram-negative bacteria was analysed for the presence of amide-bound 3-acyloxyacyl residues. It was found that such residues are constituents of all lipid A tested (Agrobacterium tumefaciens, Chromobacterium violaceum, Pseudomonas aeruginosa, Xanthomonas sinensis, Bacteroides fragilis, Vibrio cholerae, Fusobacterium nucleatum, Rhodospirillum tenue, Acinetobacter calcoaceticus, and Escherichia coli). Amide-linked (R)-3-acyloxyacyl groups, therefore, represent common and ubiquitous structural elements of bacterial lipid A. The composition of 3-acyloxyacyl groups differed considerably among different bacteria. As amide-bound (R)-3-hydroxy fatty acids straight chain and isobranched acyl groups with 10-17 carbon atoms were identified. The most frequently encountered fatty acids, substituting the 3-hydroxyl group of 3-hydroxy fatty acids, were nonhydroxylated straight chain and isobranched acyl residues with 10-17 carbon atoms as well as (S)-2-hydroxy fatty acids with 12 carbon atoms. In some cases, using laser desorption mass spectrometry, the distribution of 3-acyloxyacyl residues over the two available glucosamine amino groups of the lipid A backbone was investigated.     

Stereochemistry of biopterin cofactor and facile methods for the determination of the stereochemistry of a biologically active 5,6,7,8-tetrahydropterin

The structure of (-)-tetrahydrobiopterin, the cofactor for phenylalanine, tyrosine, and tryptophan hydroxylases, was determined as (6R, 1'R, 2'S)-6-(1',2'-dihydroxypropyl)-5,6,7,8-tetrahydropterin by X-ray crystallographic analysis. The CD spectra of (-)-tetrahydrobiopterin exhibits a negative Cotton effect at 290-240 nm in pH 3.2 solution. The relationship of the negative Cotton effect and the 6R configuration was supported by the application of CD analysis with 2-methyltetrahydronaphthalene as a model compound. The stereochemistries at the C(6) position of various biologically active 6-substituted tetrahydropterins were determined from the CD spectra. The relative configuration of two asymmetric centers, C(6), and C(1'), was determined by HPLC analysis on a strong cation exchange column.     

Identification and egg hatching activity of monohydroxy fatty acid eicosanoids in the barnacle Balanus balanoides.

Monohydroxy fatty acids (MHFAs) were isolated from homogenates of the barnacle Balanus balanoides and identified by gas chromatography-mass spectrometry (GC-MS) as 14- and 17-hydroxy docosahexaenoic acids, 8-, 11-, 12-, 15- and 18-hydroxy eicosapentaenoic acids, 13- and 16-hydroxyoctadecatrienoic acids and 9-, 13- and 15-hydroxyoctadecadienoic acids. Each monohydroxy fatty acid was tested for egg hatching activity in a bioassay using Elminius modestus egg masses, but 8-hydroxy-5, 9, 11, 14, 17-eicosapentaenoic acid (8-HEPE) was the only MHFA with barnacle egg hatching activity. Studies on the egg hatching activity of MHFAs prepared from the oxidation of polyunsaturated fatty acids showed that activity was confined to the 8-hydroxy isomer of eicosapentaenoic acid and arachidonic acid, and that unsaturation at C5 and C14, but not C17, was essential for activity. In addition, the 8(R) conformation is necessary for activity, as 8(R)-HEPE caused egg hatching at 10(-7) M whereas the enantiomer 8(S)-HEPE was inactive.     

Configuration at C-25 in 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol excreted by patients with cerebrotendinous xanthomatosis: circular dichroism and 13C-NMR studies

The configuration at C-25 in 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,25,26-pentol isolated from the bile and feces of patients with cerebrotendinous xanthomtosis (CTX) was determined from the lanthanide-induced circular dichroism (CD) Cotton effects and 13C-NMR measurements. Under anhydrous conditions, CD spectra of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,25,26-pentol in the presence of Eu(fod)3 exhibited a large induced negative Cotton effect at 320 nm. On the basis of the empirical rule (primary-tertiary-alpha-diols) in which R compounds have positive Cotton effects and S compounds have negative Cotton effects at 320 nm, it was concluded that 25,26-pentol has the 1,2,glycol structure with C-25 having the S-configuration. This assignment was based upon comparison with model compounds, 25(R and S),26-dihydroxy cholesterols and 25(R and S),26-dihydroxy cholecalciferols whose single-crystal X-ray structure and 13C-NMR studies have been performed. It is suggested that these data may be helpful to clarify the stereospecificity of the hydroxylation of the terminal methyl group of the cholesterol side chain in CTX.     

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

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

Metabolic origin of urinary 3-hydroxy dicarboxylic acids

3-Hydroxy dicarboxylic acids with chain lengths ranging from 6 to 14 carbons are excreted in human urine. The urinary excretion of these acids is increased in conditions of increased mobilization of fatty acids or inhibited fatty acid oxidation. Similar urinary profiles of 3-hydroxy dicarboxylic acids were also observed in fasting rats. The metabolic genesis of these urinary 3-hydroxy dicarboxylic acids was investigated in vitro with rat liver postmitochondrial and mitochondrial fractions. 3-Hydroxy monocarboxylic acids ranging from 3-hydroxyhexanoic acid to 3-hydroxyhexadecanoic acid were synthesized. In the rat liver postmitochondrial fraction fortified with NADPH, these 3-hydroxy fatty acids with carbon chains equal to or longer than 10 were oxidized to (omega - 1)- and omega-hydroxy metabolites as well as to the corresponding 3-hydroxy dicarboxylic acids. 3-Hydroxyhexanoic (3OHMC6) and 3-hydroxyoctanoic (3OHMC8) acids were not metabolized. Upon the addition of mitochondria together with ATP, CoA, carnitine, and MgCl2, the 3-hydroxy dicarboxylic acids were converted to 3-hydroxyoctanedioic, trans-2-hexenedioic, suberic, and adipic acids. In the urine of children with elevated 3-hydroxy dicarboxylic acid levels, 3OHMC6, 3OHMC8, 3-hydroxydecanoic, 3,10-dihydroxydecanoic, 3,9-dihydroxydecanoic, and 3,11-dihydroxydodecanoic acids were identified. On the basis of these data, we propose that the urinary 3-hydroxy dicarboxylic acids are derived from the omega-oxidation of 3-hydroxy fatty acids and the subsequent beta-oxidation of longer chain 3-hydroxy dicarboxylic acids. These urinary 3-hydroxy dicarboxylic acids are not derived from the beta-oxidation of unsubstituted dicarboxylic acids.     

Absolute Configurations of Four Resorcylic Acid Lactones,Paecilomycins J − M,by CD/TDDFT Calculations

The absolute configurations of four resorcylic acid lactones (RALs), paecilomycins J ? M ( 1 ? 3 and 5 ), were assigned by Time‐Dependent Density‐Functional Theory (TDDFT) calculations of their electronic circular dichroism (CD) spectra. The previously reported structure 4 for paecilomycin M was found to be incorrect and should be changed to structure 5 . Analysis of structure‐spectrum relationship for this group of RALs suggested that V′‐shape conformations give type I CD spectra (two negative Cotton effects around 300 and 260 nm, a positive Cotton effect around 220 nm) while V‐shape conformations yield type II spectra (signs of three Cotton effects were opposite to those in type I). Chirality 26:44–50, 2013. © 2013 Wiley Periodicals, Inc.     

Metabolism of 18:2(n - 6), 18:3(n - 3), 20:4(n - 6) and 20:5(n - 3) by the fungus Gaeumannomyces graminis: identification of metabolites formed by 8-hydroxylation and by w2 and w3 oxygenation.

The present study was aimed at developing a cell-free preparation of Gaeumannomyces graminis to biosynthesize w2-hydroxy, w3-hydroxy and related metabolites of essential fatty acids. 14C-labelled linoleic acid (18:2(n - 6)), linolenic acid (18:3(n - 3)), arachidonic acid (20:4(n - 6)) and eicosapentaenoic acid (20:5(n - 3)) were incubated with the cytosolic and microsomal fractions and NADPH. Significant metabolism was only found in the cytosol. The main products were purified by high-performance liquid chromatography and identified by gas chromatography-mass spectrometry (GC-MS). 18:2(n - 6) was metabolized mainly to 8-hydroxy-9,12-octadecadienoic acid (8-HODE), while the w2 and the w3 alcohols were formed in relatively small amounts. The absolute configuration of the 8-hydroxyl was found to be R by ozonolysis of the diastereoisomeric (-)-menthoxycarbonyl derivative of 8-HODE and GC-MS analysis. In analogy, 18:3(n - 3) was converted to 8-hydroxy-9,12,15-octadecatrienoic acid and to smaller amounts of the 15,16-diol (15,16-DiHODE). In contrast, 8-hydroxy metabolites of 20:4(n - 6) or 20:5(n - 3) could not be detected. 20:4(n - 6) was efficiently converted to 18(R)-hydroxyeicosatetraenoic acid (18(R)-HETE) and 19(R)-HETE and to traces of 17-HETE, while 20:5(n - 3) was mainly metabolized to the 17,18-diol (17,18-DiHETE) and to smaller amounts of the w2 alcohol. In conclusion, the cytosol of G. graminis can be used for stereoselective biosynthesis of some hydroxy metabolites of essential fatty acids.     

Quantification of ergosterol and 3-hydroxy fatty acids in settled house dust by gas chromatography-mass spectrometry: comparison with fungal culture and determination of endotoxin by a Limulus amebocyte lysate assay.

Ergosterol and 3-hydroxy fatty acids, chemical markers for fungal biomass and the endotoxin of gram-negative bacteria, respectively, may be useful in studies of health effects of organic dusts, including domestic house dust. This paper reports a method for the combined determination of ergosterol and 3-hydroxy fatty acids in a single dust sample and a comparison of these chemical biomarkers determined by gas chromatography-mass spectrometry with results from fungal culture and Limulus assay. Analyses of replicate house dust samples resulted in correlations of 0.91 (ergosterol in six replicates; P < 0.01) and 0.94 (3-hydroxy fatty acids in nine replicates; P < 0.001). The amounts of ergosterol (range, 2 to 16.5 ng/mg of dust) correlated with those of total culturable fungi (range, 6 to 1,400 CFU/mg of dust) in 17 samples, (r = 0.65; P < 0.005). The amounts of endotoxin (range, 11 to 243 endotoxin units/mg of dust) measured with a modified chromogenic Limulus assay correlated with those of lipopolysaccharide (LPS) determined from 3-hydroxy fatty acid analysis of 15 samples. The correlation coefficient depended on the chain lengths of 3-hydroxy acids used to compute the LPS content. The correlation was high (r = 0.88 +/- 0.01; P < 0.001) when fatty acid chains of 10 to 14 carbon atoms were included; the correlation was much lower when hydroxy acids of 16- or 18-carbon chains were included. In conclusion, the results of the described extraction and analysis procedure for ergosterol and 3-hydroxy fatty acids are reproducible, and the results can be correlated with fungal culture and endotoxin activity of organic dust samples.     

Impact of free hydroxylated and methyl-branched fatty acids on the organization of lipid membranes

Differential scanning calorimetry (DSC) has been applied to study the effect of free hydroxylated and methyl-branched fatty acids on the physico-chemical properties of lipid membranes. First, the impact of free hydroxy fatty acids (HFAs) on dimyristoylphosphatidylcholine (DMPC) model membranes was monitored only as a function of chain length and position of the attached hydroxyl group. Second, racemic vs. enantiopure anteiso fatty acids (AFAs) and HFAs were investigated to address the question of which role does a fatty acid's chirality play on its membrane pertubing effect. The DSC thermograms revealed that the main gel to liquid-crystalline phase transition of the DMPC bilayers which results in a disordering effect of the lipid hydrocarbon chains was affected in different ways depending on the nature of the incorporated fatty acid. Long-chain 2- and 3-HFAs stabilized the gel phase by reducing the phase transition temperature (T(m)), whereas short-chain HFAs and long-chain HFAs with the hydroxy group remote from the head group stabilized the more disordered liquid-crystalline state. Additionally, we observed that enantiopure (S)-14-methylhexadecanoic acid ((S)-a17:0) and (R)-2-hydroxy octadecanoic acid and the corresponding racemates had contrary effects upon incorporation into DMPC bilayers. In both cases, the pure enantiomers alleviated the liquid-crystalline state of the biological model membrane.     

Chromatographic determination of optical configuration of 3-hydroxy fatty acids composing microbial surfactants

Abstract The chromatographic determination of the optical configuration of 3-hydroxy fatty acids of microbial surfactants was achieved in chiral high pressure liquid chromatography (HPLC) by injecting 3,5-dinitroaniline-derivatives of crude hydrolysates (less than 1 mg). Serrawettin W2, a surface-active cyclodepsipeptide of Serratia marcescens , was shown to contain d -3-hydroxydecanoic acid. Rubiwettin R1 and RG1, surface active glycolipid and linked fatty acids of Serratia rubidaea , were shown to contain d -3-hydroxytetradecanoic acid and d -3-hydroxydecanoic acid. The new method does not require purified sample or authentic optical isomers, and could be useful in the structural analysis of microbial lipids.     

Configurational assignment of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23, 25-pentol excreted by patients with cerebrotendinous xanthomatosis (a circular dichroism study).

The absolute configuration of the C27 pentahydroxy bile alcohol present in bile and feces of two patients with cerebrotendinous xanthomatosis (CTX) was determined by circular dichroism (CD) spectroscopy. Under anhydrous conditions CD spectra of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23, 25-pentol in the presence of Eu (fod) 3[tris (1, 1, 1, 2, 2, 3, 3-hepta fluoro-7, 7-dimethyl-octane-4, 6-dionato) europium (III)] exhibited a large induced split Cotton effect at ca. 310 nm. From the induced circular dichroism of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23, 25-pentol with Eu(fod) 3 it was concluded that the CTX bile alcohol has the 1, 3 glycol structure with carbon 23 having the R configuration. This information will be useful in elucidating a structural mechanism for the conversion of 5 beta-cholestranepentols into bile acids in man and rat.     

Nature and linkage type of fatty acids present in lipopolysaccharides of phase I and II Coxiella burnetii

The constituent fatty acids of lipopolysaccharides (LPS) of Coxiella burnetii (phase I and II) were qualitatively and quantitatively analysed by combined gas-liquid chromatography/mass spectrometry. The total fatty acid content (per mg LPS) was determined as 90.0 nmol (2.3 wt%) for LPS of phase I cells (LPS I) and 179.1 nmol (4.8 wt%) for LPS of phase II cells (LPS II). Of the 24 different acyl residues characterized (12 to 18 carbon atoms), nine were 3-hydroxy fatty acids (normal, iso- and anteiso-branched) which quantitatively predominated. All 3-hydroxylated fatty acids were found to possess the (R)-configuration, to be exclusively amide-linked and to be acylated at their 3-hydroxyl group. Ester-linked nonhydroxylated fatty acids (normal, iso- and anteiso-branched) were present but ester-bound 3-hydroxy- or 3-acyloxyacyl residues were lacking from C. burnetii LPS I and LPS II. As the major acyl group (R)-3-(12-methyl-tetradecanoyloxy)-12-methyl-tetradecanoic acid was identified. Our results show that the complex fatty acid spectrum of C. burnetii differs considerably from that of LPS of other Gram-negative bacteria. They further suggest an enormous heterogeneity of the lipid A component of C. burnetii LPS I and LPS II.     

Sterically hindered disulfide bridges in cystine diketopiperazine, cysteinyl-cysteine disulfide and derivatives.

L-Cystine diketopiperazine (1), L-cysteinyl-cysteine disulfide -HCl (2), L-cysteinyl-cysteine disulfide methyl ester -HCl (3), and t-butyloxycarbonyl-L-cysteinyl-cysteine disulfide methyl ester (4) are investigated by CD, ultraviolet, 13C NMR, infrared and laser Raman spectroscopy. The temperature dependence of the 13C NMR signals of 1 reveals an exceptionally high energy barrier of deltaGNo. = 15.8 +/- 0.2 kcal/mol for the reversible change in helicity of the inherently dissymmetric disulfide bridge of 1. The P-helical diastereomer predominates in dimethyl-sulfoxide at 25 degrees C, with 80-85% of the molecules having this configuration. The Cotton effects of 1 are larger and show smaller temperature coefficients than the conformationally more mobile cystine compounds 2 and 3. After dissolving crystals of 1 in 95% ethanol there is a time-dependent decrease of the ellipticity of the negative Cotton effect at 225 nm, indicating a conformational change in going from crystal to solution. Besides 1, 2 and 3 are at present the only known examples of cystine derivatives with C-S-S-C torsional angles around 90 degrees, which do not exhibit optical activity in the long wavelength disulfide absorption, as is predicted for 1 from the Linderberg-Michl model. At 305 nm a new weak Cotton effect was discovered for 1. The solvent dependence of the CD spectra is discussed and the infrared and Raman spectra are assigned.     

Acyl-acyl carrier protein specificity of UDP-GlcNAc acyltransferases from gram-negative bacteria: relationship to lipid A structure.

Lipid A, the component of lipopolysaccharide that provides the membrane anchor of the core and O-antigen sugars, is known to contain characteristic R-3-hydroxy fatty acids bound to the 2,2' (N-linked) and 3,3' (O-linked) positions of the glucosamine disaccharide in different gram-negative bacteria. The studies reported here show that it is the acyl-acyl carrier protein specificities of the enzymes UDP-GlcNAc-O-acyltransferase and UDP-3-O-[(R)-3-hydroxyacyl]-GlcN-N-acyltransferase that determine the nature of these fatty acids.     

Cellular Lipid and Fatty Acid Compositions of Burkholderia pseudomallei Strains Isolated from Human and Environment in Viet Nam

Viet nam is known as an endemic area of melioidosis but its etiologic agent originated in Viet nam was not extensively studied. For the first time, we analyzed the cellular lipid and fatty acid compositions of 15 Vietnamese isolates of Burkholderia pseudomallei, 10 from humans and 5 from the environment. Cellular lipid compositions were analyzed by two-dimensional thin-layer chromatography on silica gel G plates. Cellular fatty acid methyl esters were analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The major lipids in all the isolates were phosphatidylglycerol (PG), two forms of phosphatidylethanolamine (PE-1 and PE-2), and two forms of ornithine-containing lipid (OL-1 and OL-2). PE-1 contained non-hydroxy fatty acids at both sn-1 and ?2 positions, while PE-2 possessed 2-hydroxy fatty acids and non-hydroxy fatty acids in a ratio of 1: 1. Since snake venom phospholipase A₂ digestion of PE-2 liberated 2-hydroxy fatty acids, it was confirmed that these acids are at the sn-2 position of glycerol moiety. In both OL-1 and OL-2, amide-linked fatty acid was 3-hydroxy palmitic acid (3-OH-C16: 0), while ester-linked fatty acids were non-hydroxy acids in OL-1 and 2-hydroxy acids in OL-2. The total cellular fatty acid compositions of the test strains were characterized by the presence of 2-hydroxy palmitic (2-OH-C16: 0), 2-hydroxy hexadecenoic (2-OH-C16: 1), 2-hydroxy octadecenoic (2-OH-C18: 1), 2-hydroxy methylene octadecanoic (2-OH-C19CPA), 3-hydroxy myristic (3-OH-C14: 0) and 3-hydroxy palmitic (3-OH-C16: 0) acids. There were significant differences in the concentration of hexadecenoic (C16: 1), methylene hexadecanoic (C17CPA), octadecenoic (C18: 1) and methylene octadecanoic (C19CPA) acids among the Vietnamese isolates of B. pseudomallei. However, no significant difference was observed in cellular lipid and fatty acid components between strains of human and environmental origins.     

Characterization of glucosylceramides in the Polygonaceae, Rumex obtusifolius L. injurious weed

Rumex obtusifolius L., a member of Polygonaceae, is one of the world's worst weeds. We characterized the glucosylceramide molecular species in leaves of R. obtusifolius by liquid chromatography/tandem mass spectrometry. 4,8-Sphingadienines were principally paired with 2-hydroxy palmitic acids. In contrast, 4-hydroxy-8-sphingenines were chiefly attached to 2-hydroxy fatty acids with 22 to 26 carbon-chain length. A unique characteristic of the 2-hydroxy fatty acid composition of R. obtusifolius was the high content of n-9 monoenoic 2-hydroxy fatty acids with 22 and 24 carbon-chain length. The levels of the Z and E stereoisomers of the 8-unsaturated long-chain bases were reliably distinguished from those in other plant families in ten species of Polygonaceae.     

Incomplete fatty acid oxidation. The production and epimerization of 3-hydroxy fatty acids.

3-Hydroxydicarboxylic acids are major urinary metabolites derived from fatty acid metabolism. These compounds are produced from the omega-oxidation of 3-hydroxy fatty acids. The production of the precursor 3-hydroxy fatty acids from incomplete beta-oxidation of fatty acids in rat liver mitochondria was investigated. Independent of the chain length or the concentration of fatty acid substrates, the accumulation of 3-hydroxyacyl intermediates was relatively constant at the concentration of 3-5 nmol/mg of mitochondrial protein. The extent of the incomplete oxidation was the same in Percoll gradient-purified mitochondria. Rotenone treatment increased the production of 3-hydroxy fatty acids. 3-Hydroxy fatty acids did not exist as pure L-enantiomer as expected from beta-oxidation. Instead, these metabolites were epimerized to a near racemic mixture of D- and L-isomers with a slightly dominant D-isomer (58 +/- 3%). By using deuterium-isotope labeling, the mechanism of epimerizartion was shown to be a rapid dehydration-rehydration through trans-2-enoyl-CoA. In addition, cis-3 and trans-3 fatty acids were produced; these metabolites were derived from the isomerization of trans-2-enoyl-CoA. Epimerase and isomerase were thought to be enzymes involved in the oxidation of unsaturated fatty acids. Current data have shown that the metabolism of these acids is actually through NADPH-dependent reduction pathways. The activities of epimerase and isomerase detected in rat liver mitochondria possibly function mainly in the metabolism of saturated fatty acids in a reverse role to the conventional concept.     

Novel free ceramides as components of the soldier defense gland of the Formosan subterranean termite (Coptotermes formosanus)

Of the lipid extracts of the defense secretion from the Formosan subterranean termite, Coptotermes formosanus Shiraki, on high-performance thin-layer chromatography analysis, no glycolipids or phospholipids were detected, but free fatty acids and three novel ceramides were found (termed TL-1, TL-2, and TL-3). Free fatty acids were confirmed to be lignoceric acid (C24:0) and hexacosanoic acid (C26:0), as described previously [Chen, J., G. Henderson, and R. A. Laine. 1999. Lignoceric acid and hexacosanoic acid: major components of soldier frontal gland secretions of the Formosan subterranean termite (Coptotermes formosanus). J. Chem. Ecol. 25: 817-824]. TL-1, TL-2, and TL-3 were characterized as ceramides differing in hydrophobicity based on results of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry analysis, mild alkaline treatment, GC-MS analysis of fatty acid methylesters, and GC-MS analysis of sphingoid long-chain bases (LCBs) as trimethylsilyl derivatives. Fatty acids in TL-1 and TL-2 were C18:0, C20:0, and C22:0, and those in TL-3 were 2-hydroxy C18:0, C20:0, and C22:0. The most predominant LCB in TL-2 was a novel trihydroxy C(14)-sphingosine, 1,3,9-trihydroxy-2-amino-6-tetradecene. TL-3 contained C(18)-sphinganine and two kinds of novel sphingadienines, 1,3-dihydroxy-2-amino-7,10-hexadecadiene and 1,3-dihydroxy-2-amino-11,14-eicosadiene. Although examination of the biological activities of these novel ceramides was beyond the scope of these studies, because of the minuscule quantities available from termite secretions, it will be interesting in the future to synthesize these molecules for biological testing.