Alkylpyrazines: alarm pheromone components of the little fire ant, Wasmannia auropunctata (Roger) (Hymenoptera, Formicidae)

The previous identification of 2,5-dimethyl-3-(3-methylbutyl)pyrazine as the mandibular alarm pheromone of the little fire ant Wasmannia auropunctata (Roger), has been found to be incorrect. Gas chromatography–mass spectrometry (GC/MS) of ant extracts suggested the correct structure to be the regioisomer 2,5-dimethyl-3-(2-methylbutyl)pyrazine, which was confirmed by comparison with the synthetic pyrazine. GC/MS analysis also revealed the presence of an additional disubstituted alkylpyrazine which was identified as 3-methyl-2-(2-methylbutyl)pyrazine. Headspace sampling of confined ants with SPME and Porapak Q followed by GC/MS analysis showed 2,5-dimethyl-3-(2-methylbutyl)pyrazine as the major volatile released by W. auropunctata workers while 3-methyl-2-(2-methylbutyl)pyrazine was only detected in trace amounts. In laboratory bioassays, W. auropunctata workers were attracted and arrested by both pyrazines, although the results were not always consistent. Synthetic pyrazines generally attracted as many W. auropunctata workers as were attracted to a single crushed ant. However, higher numbers of W. auropunctata were arrested by crushed ant treatments than by synthetic pyrazines in all bioassays but one.     

Ecdysteroid hormones and metabolites of the stone crab, Menippe mercenaria

The Y-organs of the xanthid crab Menippe mercenaria secrete the ecdysteroids, 3-dehydroecdysone (3DE) and lesser amounts of 3-dehydro (or 2-dehydro)-25-deoxyecdysone (3D25dE) in vitro. These ecdysteroids were identified by elution-time comparisons with authentic standards, mass spectrography, and, for 3D25dE, infrared spectrometry. Tissues were incubated 18 hr with [(3)H]3DE. Activities representing 3beta-reductase and 20-hydroxylase generally were present, evidenced by finding in the tissue/medium extract labeled ecdysone (E) and 20-hydroxyecdysone (20E). Labeled 3-dehydro-20-hydroxyecdysone (3D20E) also appeared to be present. Tissue blanks and hemolymph were devoid of activity. Muscle was low, hypodermis was intermediate, and hindgut and gonads were high in activity of the enzymes. Consistent with the presence of these enzymes in peripheral tissues, ecdysteroid products identified in the hemolymph were 20E, 3D20E, and 25-deoxy-20-hydroxyecdysone (25d20E; ponasterone A). Structures of 20E and 3D20E were confirmed by co-elution with authentic standards in high-performance liquid chromatography (HPLC), co-elution of derivatives in gas chromatography, and mass spectroscopy. Ponasterone A (identified by HPLC co-elution with the standard), like 20E is present in the hemolymph in prominent amounts. These data indicate that Menippe, among crustaceans thus far studied, secretes a unique combination of ecdysteroid hormones, namely, a 3- (or 2-) oxo compound and a 25-deoxy compound. This represents a different kind of branch point from 5beta-diketol in ecdysteroid biosynthesis, in which the intermediate, 5beta-ketodiol is bypassed. A result is the joint appearance in the circulation of the hormones, 20E and ponasterone A, which in other species are singly prominent.     

Separation of some natural and synthetic corticosteroids in biological fluids and tissues by high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) technique was developed for the determination of radiolabeled triamcinolone acetonide (TAC), cortisol and their metabolites in rhesus monkey plasma, urine and tissue samples. After protein precipitation, the parent compounds and metabolites were simultaneously resolved using a single-column reversed-phase HPLC system. TAC was subsequently verified by mass spectrometry and TAC glucuronide was tentatively identified by enzymatic hydrolysis and mass spectrometry of the hydrolysis product. The endogenous hormones, cortisol and cortisone were presumptively identified by cochromatography with authentic standards on two different HPLC systems and positively identified by reverse-isotope recrystallization. Other metabolites of both compounds were detected by selective enzymatic hydrolysis and HPLC. This method is rapid and reproducible with a total recovery > 80%.     

Pyrazines as warning odour components in the Monarch butterfly, Danaus plexippus, and in moths of the genera Zygaena and Amata (Lepidoptera)

2-Methoxy-3-alkylpyrazines have been identified as odour components in Danaus plexippus, Zygaena lonicerae and an Australian Amata species, by means of coupled gas chromatography/mass spectrometry, with selected ion monitoring. Wide variability in pyrazine content was observed with D. plexippus and this was correlated with similar variability in the larval food-plants, Asclepias spp., which are presumed to be the odour sources for this butterfly. The importance of pyrazines in the biology of aposematic insects and their role as ingredients of a generalized warning system are discussed.     

Identification of YH439 and its metabolites in rat urine by gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry

YH439 is a potential drug candidate for the treatment of various hepatic disorders. YH439 and its three metabolites have been identified in rat urine by liquid chromatography–mass spectrometry (LC–MS) and by gas chromatography (GC)–MS. Identification of YH439 and its metabolites was established by comparing their GC retention times and mass spectra with those of the synthesized authentic standards. Both electron impact- and positive chemical ionization MS have been evaluated. The metabolism study was performed in the rat using oral administration of the drug. A major metabolite (YH438) was identified as the N-dealkylation product of YH439. Other identified metabolites were caused by the loss of the methyl thiazolyl amine group (metabolite II) from YH439, the isopropyl hydrogen malonate group (metabolite IV) and the decarboxylated product (metabolite III) of metabolite II.     

Analysis of bile acid glutathione thioesters by liquid chromatography/electrospray ionization-tandem mass spectrometry

The formation of thioester-linked glutathione (GSH) conjugates of bile acids (BAs) is presumed to occur via trans-acylation reactions between GSH and reactive acyl-linked metabolites of BAs. The present study examines the chemical reactivity of cholyl-adenylate and cholyl-CoA thioester, acyl-linked metabolites of cholic acid (CA), with GSH to form CA-GSH conjugate in vitro. The authentic specimen of CA-GSH was synthesized along with GSH conjugates of four common BAs found in the human body. Their structures were confirmed by proton-nuclear magnetic resonance spectroscopy and electrospray ionization (ESI)-tandem mass spectrometry in positive- and negative-ion modes. Incubation of cholyl-adenylate or cholyl-CoA thioester with GSH was carried out at pH 7.5 and 37 degrees C for 30 min, with analysis of the reaction mixture by liquid chromatography/ESI-tandem mass spectrometry, where CA-GSH was detected on the product ion mass chromatograms monitored with stable and abundant dehydrated positive-ion [M+HH(2)O](+) at m/z 680.3 and fragmented negative-ion [GSHH](-) at m/z 306.0, and was definitely identified by CID spectra by comparison with those of the authentic sample. The results show that both cholyl-adenylate and cholyl-CoA thioester are able to acylate GSH in vitro.     

Degradation of phenanthrene and anthracene by cell suspensions of Mycobacterium sp. strain PYR-1

Cultures of Mycobacterium sp. strain PYR-1 were dosed with anthracene or phenanthrene and after 14 days of incubation had degraded 92 and 90% of the added anthracene and phenanthrene, respectively. The metabolites were extracted and identified by UV-visible light absorption, high-pressure liquid chromatography retention times, mass spectrometry, (1)H and (13)C nuclear magnetic resonance spectrometry, and comparison to authentic compounds and literature data. Neutral-pH ethyl acetate extracts from anthracene-incubated cells showed four metabolites, identified as cis-1,2-dihydroxy-1,2-dihydroanthracene, 6,7-benzocoumarin, 1-methoxy-2-hydroxyanthracene, and 9,10-anthraquinone. A novel anthracene ring fission product was isolated from acidified culture media and was identified as 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid. 6,7-Benzocoumarin was also found in that extract. When Mycobacterium sp. strain PYR-1 was grown in the presence of phenanthrene, three neutral metabolites were identified as cis- and trans-9,10-dihydroxy-9,10-dihydrophenanthrene and cis-3,4-dihydroxy-3,4-dihydrophenanthrene. Phenanthrene ring fission products, isolated from acid extracts, were identified as 2,2'-diphenic acid, 1-hydroxynaphthoic acid, and phthalic acid. The data point to the existence, next to already known routes for both gram-negative and gram-positive bacteria, of alternative pathways that might be due to the presence of different dioxygenases or to a relaxed specificity of the same dioxygenase for initial attack on polycyclic aromatic hydrocarbons.     

Metabolism of arachidonic acid by human nasal and bronchial epithelial cells

Nasal and bronchial epithelium from normal human nasal turbinates was isolated from surgical specimens and used to study arachidonic acid metabolism. High-performance liquid chromatography analysis of cell incubations in the presence of calcium ionophore, A23187, showed the formation of 15-lipoxygenase products. The major arachidonic acid metabolite with bronchial and nasal tissue was 15-HETE identified by uv spectroscopy, coelution with the authentic standards by HPLC, and GC-mass spectrometry. The second major metabolite, formed from either arachidonic acid or 15-HPETE, was identified as 13-hydroxy-14,15-epoxy-5,8,11-eicosatetraenoic acid (15-alpha-HEPA) by uv spectroscopy, coelution with the authentic standard, and GC-mass spectrometry. In addition, two 8,15-diHETEs and two 8,15-LTs were identified by uv spectroscopy and coelution with the authentic standards by HPLC on both reverse-phase and normal-phase HPLC. Also isolated and identified were 14,15-diHETEs, and 12-HETE. Nasal epithelial cells appear to be more active than nasal bronchial cells in oxidizing arachidonic acid. However, the profile of metabolites from these normal tissue preparations was similar. The addition of 15-lipoxygenase products to nasal epithelium weakly stimulated Cl- ion secretion. These studies indicate that human pulmonary epithelial cells selectively oxidize arachidonic acid to 15-lipoxygenase metabolites.     

Leukotriene E4 elimination and metabolism in normal human subjects

Radiolabeled leukotriene (LT) E4 was infused into three healthy subjects in order to assess the production and elimination of sulfidopeptide leukotriene metabolites in urine. Three different radiolabeled tracers were employed, [14,15-3H]LTE4, [35S]LTE4, and [14C] LTE4 in five separate infusion studies. There was a rapid disappearance of radioactivity from the vascular compartment in an apparent two-phase process. The first elimination phase had an apparent half-life of approximately 7 min. Radioactivity quickly appeared in the urine with 10-16% eliminated during the first 2 h following intravenous infusion; 7%, 2-5 h; 4%, 5-8 h; 4%, 8-15 h; and 1.5%, 15-24 h from the [14C] LTE4 experiments. Unmetabolized LTE4 was the major radioactive component in the first urine collection, but at later times two more polar compounds predominated. After extensive purification by normal phase-solid phase extraction and reverse-phase high performance liquid chromatography, these compounds were characterized by UV spectroscopy, co-elution with synthetic standards, negative ion electron capture gas chromatography/mass spectrometry, and tandem mass spectrometry. The two major urinary metabolites were structurally determined to be 14-carboxy-hexanor-LTE3 and the conjugated tetraene, 16-carboxy-delta 13-tetranor-LTE4. Three other minor metabolites were detectable in the first urine collection only and were characterized by co-elution with synthetic standards as 16-carboxy-tetranor-LTE3, 18-carboxy-dinor-LTE4, and 20-carboxy-LTE4. omega-Oxidation and subsequent beta-oxidation from the methyl terminus appeared to be the major metabolic fate for sulfidopeptide leukotrienes in man. The accumulation of the 14-COOH-LTE3 and 16-COOH-delta 13-LTE4 may reflect a rate-limiting step in further oxidation of these compounds which places a conjugated triene or conjugated tetraene, respectively, two carbons removed from the CoA ester moiety. Also in the first urine collection there was another minor metabolite identified as N-acetyl-LTE4, however, no subsequent beta-oxidation of this metabolite was observed. The major metabolites of LTE4 might be useful in assessing in vivo production of sulfidopeptide leukotrienes in humans.     

Metabolism of the soy isoflavones daidzein, genistein and glycitein in human subjects. Identification of new metabolites having an intact isoflavonoid skeleton

Epidemiological studies have associated high soy intake with a lowered risk for certain hormone-dependent diseases. Soy and soy foods are rich sources of isoflavones, which have been shown to possess several biological activities. In this study, the metabolism of soy isoflavones daidzein, genistein and glycitein was investigated in human subjects. The aim was to find and identify urinary phase I metabolites of isoflavones, which have an intact isoflavonoid skeleton, and which might possess some bioactivity. Six volunteers included three soy bars per day into their normal western diet for a 2-week period. Daily urine samples were collected before, and after the supplementation period. Urine samples were hydrolyzed with Helix pomatia, extracted with diethyl ether, purified with Sephadex LH-20 chromatography, and analyzed as trimethylsilyl derivatives using gas chromatography–mass spectrometry (GC–MS). The structures of the isoflavone metabolites were identified using authentic reference compounds. The metabolites, for which authentic reference compounds were not available, were identified by the interpretation of mass spectra. Several new isoflavone metabolites were identified, and the presence of previously reported metabolites confirmed. The metabolic pathways of daidzein, genistein and glycitein are presented on the basis of the identification of the metabolites in human urine after soy supplementation.     

Volatile Components of Roasted Almonds: Basic Fraction

Roasted almond volatiles were isolated by vacuum carbon dioxide distillation of acetone extracts and separated into basic and non-basic fractions. The basic fraction was analyzed by combination gas chromatography-mass spectrometry utilizing open tubular column. Seventeen pyrazines and 2-formyl pyrrole were identified by comparison of their mass spectra with reference spectra. Besides them, five compounds were tentatively identified by mass spectrometry.     

Pyrazines: occurrence, formation and biodegradation

Pyrazines are a class of compounds that occur almost ubiquitously in nature. Pyrazines can be synthesised chemically or biologically, and are used as flavouring additives. The major formation of pyrazines occurs during heating of food. There is very little information available on the degradation of these compounds. In humans and animals, pyrazines are excreted as glucuronates or bound to glutathione via the kidney after hydroxylation, but the pyrazine ring is not cleaved. Bacteria have been isolated, which are able to use various substituted pyrazines as a sole carbon and energy source. In a few cases, the initial metabolites have been characterised; however, the mechanism of ring cleavage and the further degradation pathways are still unknown and await further investigation.     

Indole-3-butyric acid (IBA) production in culture medium by wild strain Azospirillum brasilense

Some microorganisms found in the soil are able to produce substances which regulate plant growth. In this study, we show the presence of a substance associated with auxin activity, identified as indole-3-butyric acid (IBA), in Azospirillum brasilense UAP 154 growth medium. A. brasilense was grown and indolic compounds were extracted from the supernatant. These were then analyzed by high performance liquid chromatography (HPLC), gas chromatography and gas chromatography mass spectrometry. The retention time was similar to those of the authentic IBA standard. The compound obtained from HPLC was collected and applied to maize seedlings (Zea mays), inducing biological activity along the roots, similar to that induced by an authentic IBA standard.     

Occurrence of 2-keto-deoxyoctonic acid 5-phosphate in lipopolysaccharides of Vibrio cholerae Ogawa and Inaba.

A phosphorylated 2-keto-3-deoxyoctonic acid (KDO) was released from the lipopolysaccharides of Vibrio cholerae Ogawa and Inaba after strong acid hydrolysis. The phosphorylated KDO was identified by gas-liquid chromatography and mass spectrometry after reduction and permethylation as KDO-5-phosphate and an isomer of it being phosphorylated at position 7 or 8. After treatment with alkaline phosphatase, KDO was detected by gas-liquid chromatography and mass spectrometry. It was indistinguishable from authentic 2-keto-3-deoxy-D-manno-octonic acid.     

Isolation and Characterization of a Dibenzofuran-Degrading Yeast: Identification of Oxidation and Ring Cleavage Products

We characterized the ability of a yeast to cleave the aromatic structure of the dioxin-like compound dibenzofuran. The yeast strain was isolated from a dioxin-contaminated soil sample and identified as Trichosporon mucoides. During incubation of glucose-pregrown cells with dibenzofuran, six major metabolites were detected by high-performance liquid chromatography. The formation of four different monohydroxylated dibenzofurans was proven by comparison of analytical data (gas chromatography-mass spectrometry) with that for authentic standards. Further oxidation produced 2,3-dihydroxydibenzofuran and its ring cleavage product 2-(1-carboxy methylidene)-2,3-dihydrobenzo[b]furanylidene glycolic acid, which were characterized by mass spectrometry and ¹H nuclear magnetic resonance spectroscopy. These two metabolites are derived from 2-hydroxydibenzofuran and 3-hydroxydibenzofuran, as shown by incubation experiments using these monohydroxylated dibenzofurans as substrates.     

Identification and biological activity of dihydroleukotriene B4: a prominent metabolite of leukotriene B4 in the human lung

Exogenous [3H]leukotriene B4 (LTB4) was converted into several polar and non-polar metabolites in the chopped human lung. One of the major metabolites was identified as 5(S),12-dihydroxy-6,8,14-eicosatrienoic acid (10,11-dihydro-LTB4) by means of co-chromatography with authentic standards, ultraviolet spectrometry and gas chromatography-mass spectrometry. Analysis of chiral straight phase HPLC revealed the presence of both the 12(S) and 12(R) epimers of dihydro-LTB4. Dihydro-LTB4 was also formed from endogenously generated LTB4 in ionophore A23187 stimulated incubations. The dihydro metabolites were approximately 100 times less potent than LTB4 in causing guinea pig lung strip contraction and leukocyte-dependent inflammation in the hamster cheek pouch in vivo.     

Radiation metabolomics. 4. UPLC-ESI-QTOFMS-Based metabolomics for urinary biomarker discovery in gamma-irradiated rats

Radiation metabolomics has aided in the identification of a number of biomarkers in cells and mice by ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) and in rats by gas chromatography-coupled mass spectrometry (GCMS). These markers have been shown to be both dose- and time-dependent. Here UPLC-ESI-QTOFMS was used to analyze rat urine samples taken from 12 rats over 7 days; they were either sham-irradiated or γ-irradiated with 3 Gy after 4 days of metabolic cage acclimatization. Using multivariate data analysis, nine urinary biomarkers of γ radiation in rats were identified, including a novel mammalian metabolite, N-acetyltaurine. These upregulated urinary biomarkers were confirmed through tandem mass spectrometry and comparisons with authentic standards. They include thymidine, 2'-deoxyuridine, 2'deoxyxanthosine, N(1)-acetylspermidine, N-acetylglucosamine/galactosamine-6-sulfate, N-acetyltaurine, N-hexanoylglycine, taurine and, tentatively, isethionic acid. Of these metabolites, 2'-deoxyuridine and thymidine were previously identified in the rat by GCMS (observed as uridine and thymine) and in the mouse by UPLC-ESI-QTOFMS. 2'Deoxyxanthosine, taurine and N-hexanoylglycine were also seen in the mouse by UPLC-ESI-QTOFMS. These are now unequivocal cross-species biomarkers for ionizing radiation exposure. Downregulated biomarkers were shown to be related to food deprivation and starvation mechanisms. The UPLC-ESI-QTOFMS approach has aided in the advance for finding common biomarkers of ionizing radiation exposure.     

Metabolite profiling of Arabidopsis seedlings in response to exogenous sinalbin and sulfur deficiency

In order to determine how plant uptake of a sulfur-rich secondary metabolite, sinalbin, affects the metabolic profile of sulfur-deficient plants, gas chromatography time-of-flight mass spectrometry (GC-TOF-MS), in combination with liquid chromatography-mass spectrometry (LC-MS), was used to survey the metabolome of Arabidopsis seedlings grown in nutrient media under different sulfur conditions. The growth media had either sufficient inorganic sulfur for normal plant growth or insufficient inorganic sulfur in the presence or absence of supplementation with organic sulfur in the form of sinalbin (p-hydroxybenzylglucosinolate). A total of 90 metabolites were identified by GC-TOF-MS and their levels were compared across the three treatments. Of the identified compounds, 21 showed similar responses in plants that were either sulfur deficient or sinalbin supplemented compared to sulfur-sufficient plants, while 12 metabolites differed in abundance only in sulfur-deficient plants. Twelve metabolites accumulated to higher levels in sinalbin-supplemented than in the sulfur-sufficient plants. Secondary metabolites such as flavonol conjugates, sinapinic acid esters and glucosinolates, were identified by LC-MS and their corresponding mass fragmentation patterns were determined. Under sinalbin-supplemented conditions, sinalbin was taken up by Arabidopsis and contributed to the endogenous formation of glucosinolates. Additionally, levels of flavonol glycosides and sinapinic acid esters increased while levels of flavonol diglycosides with glucose attached to the 3-position were reduced. The exogenously administered sinalbin resulted in inhibition of root and hypocotyl growth and markedly influenced metabolite profiles, compared to control and sulfur-deficient plants. These results indicate that, under sulfur deficient conditions, glucosinolates can be a sulfur source for plants. This investigation defines an opportunity to elucidate the mechanism of glucosinolate degradation in vivo.     

Metabolites formed during anaerobic transformation of toluene and o-xylene and their proposed relationship to the initial steps of toluene mineralization.

Strain T1 is a facultative bacterium that is capable of anaerobic toluene degradation under denitrifying conditions. While 80% of the carbon from toluene is either oxidized to carbon dioxide or assimilated into cellular carbon, a significant portion of the remainder is transformed into two dead-end metabolites. These metabolites were produced simultaneous to the mineralization of toluene and were identified as benzylsuccinic acid and benzylfumaric acid. Identification was based on comparison of mass spectra of the methyl esters of the metabolites and authentic compounds that were chemically synthesized. Strain T1 is also capable of o-xylene transformation during growth on toluene. o-Xylene does not serve as a source of carbon and is not mineralized. Rather, it is transformed to analogous dead-end metabolites, (2-methylbenzyl)-succinic acid and (2-methylbenzyl)-fumaric acid. o-Xylene transformation also occurred during growth on succinic acid, which suggests that attack of the methyl group by succinyl-coenzyme A is a key reaction in this transformation. We reason that the main pathway for toluene oxidation to carbon dioxide involves a mechanism similar to that for the formation of the metabolites and involves an attack of the methyl group of toluene by acetyl-coenzyme A.     

Metabolites formed during anaerobic transformation of toluene and o-xylene and their proposed relationship to the initial steps of toluene mineralization.

Strain T1 is a facultative bacterium that is capable of anaerobic toluene degradation under denitrifying conditions. While 80% of the carbon from toluene is either oxidized to carbon dioxide or assimilated into cellular carbon, a significant portion of the remainder is transformed into two dead-end metabolites. These metabolites were produced simultaneous to the mineralization of toluene and were identified as benzylsuccinic acid and benzylfumaric acid. Identification was based on comparison of mass spectra of the methyl esters of the metabolites and authentic compounds that were chemically synthesized. Strain T1 is also capable of o-xylene transformation during growth on toluene. o-Xylene does not serve as a source of carbon and is not mineralized. Rather, it is transformed to analogous dead-end metabolites, (2-methylbenzyl)-succinic acid and (2-methylbenzyl)-fumaric acid. o-Xylene transformation also occurred during growth on succinic acid, which suggests that attack of the methyl group by succinyl-coenzyme A is a key reaction in this transformation. We reason that the main pathway for toluene oxidation to carbon dioxide involves a mechanism similar to that for the formation of the metabolites and involves an attack of the methyl group of toluene by acetyl-coenzyme A.