Distribution of C22-, C24- and C26-α-Unit-Containing Mycolic Acid Homologues in Mycobacteria

There are three mycolic acid homologues with C₂₂-, C₂₄- and C₂₆-α-units in Mycobacterium. In order to reveal the composition and distribution of these homologues in each subclass and molecular species of mycolic acids and to compare them with the composition of constitutive non-polar fatty acids (free and bound forms), we have separated non-polar fatty acids and each subclass of mycolic acids from 21 mycobacterial species by thin-layer chromatography, and analyzed non-polar fatty acid methyl esters by gas chromatography (GC) and the cleavage products of methyl mycolate by pyrolysis GC. We further performed mass chromatographic analysis of trimethylsilyl (TMS) ether derivatives of mycolic acid methyl esters by monitoring [B-29]⁺ ions (loss of CHO from the α-branched-chain structure of mycolic acids) of m/z 426, 454 and 482 which are attributed to C₂₂-, C₂₄- and C₂₆-α-units of TMS ether derivatives of methyl mycolates, respectively, (Kaneda, K. et al, J. Clin. Microbiol. 24: 1060-1070, 1986). By pyrolysis GC, C_22:0, C_24:0 and C_26:0 fatty acid methyl esters generated by the C₂-C₃ cleavage of C₂₂-, C₂₄- and C₂₆-α-unit-containing mycolic acid methyl esters, respectively, were detected. Their proportion was almost the same among subclasses of mycolic acids in every Mycobacterium and also similar to the proportion of constitutive non-polar C_22:0, C_24:0 and C_26:0 fatty acids. By mass chromatography, the composition and distribution of C₂₂- and C₂₄-α-unit-containing homologues were revealed to be similar between α- and α'-mycolic acids in every Mycobacterium. We further analyzed in detail M. vaccae and demonstrated that the mass chromatogram of C₂₂-α-unit-containing homologue was analogous in shape to that of the C₂₄-α-unit-containing one, with the latter mass chromatogram being up-shifted from the former by two carbon numbers, in every subclass of α-, α'-, keto and dicarboxy mycolic acids. The present study suggests that the compositions of three homologues of both mycolic acids and constitutive non-polar fatty acids, which are characteristic to each mycobacterial species, may reflect the proportion of the amount of free C_22:0, C_24:0 and C_26:0 fatty acids synthesized in the cell. It is further demonstrated that intermolecular condensation of two fatty acids which become α- and β-units of mycolic acids will occur independently of the carbon chain length or kinds of polar moieties of fatty acids.     

Characterization of lipoxins by combined gas chromatography and electron-capture negative ion chemical ionization mass spectrometry: formation of lipoxin A4 by stimulated human whole blood

The lipoxins are a recent addition to the family of bioactive products derived from arachidonic acid. Here, we have prepared pentafluorobenzyl ester, trimethylsilyl ether derivatives of lipoxin A4, lipoxin B4 and pentadeuterolipoxin A4 and have characterized these products by electron-capture negative ion chemical ionization gas chromatography/mass spectrometry (NICI GC/MS). Lipoxin A4 (5S,6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosa-tetraenoic acid; LXA4) was quantified following extraction from whole blood by stable isotopic dilution utilizing deuterium-labeled LXA4 as internal standard and selected ion monitoring of the [M--pentafluorobenzyl] anions. Studies with a second tritiated internal standard (e.g. [11,12-3H]LXA4) also showed that the recovery of LXA4 was greater than 80% following solid-phase extraction from whole blood, and greater than 90% from isolated cells. In addition, neither isolated neutrophils nor platelets oxidatively metabolized [11,12-3H]LXA4 when incubated in the presence or absence of stimuli. Whole blood incubated with either the ionophore of divalent cations (A23187), thrombin, or thrombin plus the chemotactic peptide formylmethionyl-leucine-phenylalanine generated both LXA4 and thromboxane, which were quantified by stable isotope dilution. The ratio of thromboxane to LXA4 formed by stimulated whole blood ranged from approximately 2:1 to 20:1. These results indicate that the lipoxins display suitable characteristics as their respective pentafluorobenzyl ester, trimethylsilyl ether derivatives for quantification by electron-capture NICI GC/MS. Moreover, they provide evidence that LXA4 can be generated from endogenous sources in whole blood following exposure to physiologically relevant stimuli.     

Measurement of the cortisol production rate in two sisters with 17 alpha-hydroxylase deficiency using [1,2,3,4-13C]cortisol and isotope dilution mass spectrometry

[1,2,3,4-13C]cortisol was i.v. administered to two sisters aged 11 yr (patient I) and 3 yr (patient II) who suffer from 17 alpha-hydroxylase deficiency. This is the first time that the cortisol production rate (CPR) in patients with 17 alpha-hydroxylase deficiency has been measured with a stable labelled tracer using the urinary method. The urine was collected for 3 days. High-performance liquid chromatography (HPLC) of approximately 100 ml urine extracts was carried out to isolate the small amount of cortisol metabolites excreted. The cortisol metabolites were oxidized to 11-oxo-aetiocholanolone. The isotope dilution in the methyl oxime tert-butyldimethylsilyl ether derivatives was measured by selected ion monitoring gas chromatography/mass spectrometry (GC/MS). The CPR calculated from tetrahydrocortisone (THE) and the cortolones was 765 and 536 nmol/day, respectively in patient I. The CPR in patient II was only calculated from THE and was 62 nmol/day. If radioactive labelled cortisol had been used, much larger quantities of urine would have been needed for isolation of sufficient mass of metabolites, even then purification may have been difficult. Steroid profiling of 1 ml urine samples by GC and identification by GC/MS revealed high concentrations of pregnenolone, progesterone, 11 beta-hydroxy progesterone and corticosterone metabolites. Tetrahydrocorticosterone and 5 alpha-tetrahydrocorticosterone were found in urine at elevated excretions of 2.5 and 5.7, 0.9 and 2.0 mumols/24 h, in patients I and II respectively. No cortisol metabolites were detected by routine GC or GC/MS as the low amounts excreted co-eluted with the relatively abundant corticosterone metabolites.     

Mass spectra of prostaglandins. IV. Trimethylsilyl derivatives of prostaglandins of the F series

The mass spectra of the trimethylsilyl ester trimethylsilyl ether derivatives of prostaglandins F_1α, F_2α and F_2β are reported and discussed. Accurate masses from the high resolution spectra of these compounds are also presented. These spectra are interpreted with the aid of those of the corresponding d₉-trimethylsilyl derivatives and selectively labeled trimethylsilyl ester-d₉-trimethylsilyl ether derivatives. It was found that metastabledefocusing was helpful in elucidation of the mechanisms of formation of some ions.     

Analyses of steroids in saliva using highly selective mass spectrometric techniques

Highly selective techniques of gas chromatography mass spectrometry have been used in the unequivocal identification of salivary steroids at concentrations ranging from 20 pg ml-1 to 20 ng ml-1. Oestradiol-17 beta, for example, has been identified in pregnancy saliva by gas chromatography high resolution mass spectrometry selected ion monitoring of the bis-TMS ether and by gas chromatography mass spectrometry metastable peak monitoring of the bis-tert-butyldimethylsilyl ether. Dehydroepiandrosterone sulphate has been identified in saliva, following enzymic hydrolysis, by gas chromatography high resolution mass spectrometry selected ion monitoring of the tert-butyldimethylsilyl ether and methyloxime tert-butyldimethylsilyl ether. These initial analyses have been designed to guide the development of routine immunoassay procedures which may subsequently be validated by comparison with reference gas chromatographic mass spectrometric methods.     

Identification and quantitation of 16α-hydroxy c21 steroid sulphates in plasma from pregnant women

Plasma steroids from 11 women in the last trimester of pregnancy were separated into conjugate class by chromatography on Sephadex LH-20. Fractions corresponding to mono- and disulphate conjugates were solvolyzed and the free steroids were separated by lipophilic gel chromatography into groups containing mono-, di- and trihydroxy steroids, respectively. Trimethylsilyl ether derivatives were prepared and repetitive-scanning gas chromatography-mass spectrometry (GC-MS) was used to characterize and quantitate the following 16α-hydroxy steroids in the monosulphate fraction: 3α,16α:-dihydroxy-5α-pregnan-20-one (range 8–80 ng/ml), 3β,16α-dihydroxy-5α-pregnan-20-one (6–55 ng/ml), 5α-pregnane-3α,16α,20α-triol (9–31 ng/ml) and 5α-pregnane-3β,16α,20α-triol (11–77 ng/ml). These compounds were also present as disulphates although at 5–50 times lower concentrations.     

Routine screening and quantitation of urinary corticosteroids using bench-top gas chromatography—mass-selective detection

A method was developed for the routine screening, confirmation and quantitation of corticosteroids in human urine using bench top capillary gas chromatography (GC)—mass-selective detection. The free and conjugated corticosteroid fractions were isolated by liquid—liquid partition. After evaporation to dryness under vacuum the corticosteroid residues were derivatized to form the methyloxime trimethylsilyl ether derivatives. Both GC retention data and characteristic spectral data based on authentic reference standards were used for the identification and quantitation of cortisol, cortisone, tetrahydrocortisol and tetrahydrocortisone in the ppb (ng/ml) concentration range. The method is simpler and more efficient than the other GC—mass spectrometric (MS) techniques. It is also more sensitive than the liquid chromatographic—MS method.     

Harman in human platelets.

The β-carbolines present in human platelets have been extracted with diethyl ether, isolated by liquid column chromatography and thin-layer chromatography (TLC), and identified by ultraviolet fluorimetry, gas liquid chromatography (GC) and mass spectrometry (MS). Harman (1-methyl-β-carboline) was the only β-carboline unequivocally identified in platelet samples with these techniques. Since harman is thought to be biosynthesized by the condensation of tryptamine and acetaldehyde, its formation may be of importance in the metabolism and the pharmacological-toxicological actions of alcohol.     

Gas-liquid chromatography-mass spectrometry of methylated and deuteriomethylated per-O-acetyl-aldononitriles from D-mannose

Peracetylated aldononitriles of the tetra-, tri-, and di-methyl ethers of D-mannopyranose were separated by gas-liquid chromatography, and analyzed by mass spectrometry. Through introduction of deuteriomethyl ether groups, various fragmentions constituting the mass spectra were identified and related to the parent methylated sugar structures. Also identified were several characteristic series of fragment ions that are common to two or more methylated D-mannopyranosides. As expected, mass spectra of the D-mannose derivatives were identical to those previously observed for D-glucose methylated in the same positions. Distinctive mass spectra were also recorded for all additional di-O-methyl-D-mannose derivatives. This information permits use of peracetylated aldononitrite derivatives in methylation-fragmentation analysis of aldohexans.     

Degradation of diphenylether by Pseudomonas cepacia

The microbial degradation of hard coal implies the cleavage of diaryl ether linkages in the coal macromolecule. We investigated the biodegradation of diphenylether as a model compound representing this substructure of coal. A bacterial strain isolated from soil and identified as Pseudomonas cepacia, was able to grow with diphenylether as sole source of carbon. During microbial growth, three metabolites were detected in the culture supernatant by high pressure liquid chromatography. As product of ring hydroxylation and subsequent rearomatization, 2,3-dihydroxydiphenylether was identified by UV, mass and nuclear magnetic resonance spectrometry and gas chromatography analyses. The cleavage of the ether linkage led to the formation of phenol and 2-pyrone-6-carboxylic acid, the latter being not further degraded by Pseudomonas cepacia. The possible cleavage mechanism of the ether linkage is discussed.Non-standard abbreviations DPE diphenylether - PCA 2-pyrone-6-carboxylic acid - GC gas chromatography - MS mass spectrometry - HPLC high pressure liquid chromatography     

Study of reactions induced by hydroxylamine treatment of esters of organic acids and of 3-ketoacids: application to the study of urines from patients under valproate therapy

Hydroxylamine used at alkaline pH as oximating agent in the search for organic aciduria by gas chromatography/mass spectrometry (GC/MS) induces other chemical reactions. Esters are partially transformed in their corresponding hydroxamic acids. GC/MS characteristics of the trimethylsilylated derivatives of the hydroxamic acids arising from alpha-unsaturated esters are here reported. Their mass spectral fragmentation helps in the recognition of peaks arising from the glucuronides of 2-ene- and probably 2,3'-diene-valproic acid. By heating in the injection port of the gas chromatograph, part of some trimethylsilylated hydroxamic acids are transformed to the corresponding isocyanates by a Lossen-like rearrangement. In addition to the corresponding hydroxamic acids, hydroxylamine treatment of alpha-unsaturated esters forms 2-isoxazolidin-3-ones by intramolecular Michael addition. GC/MS characteristics of the trimethylsilylated derivatives of these compounds are reported. Submitted to hydroxylamine, 3-ketoacids forms 2-isoxazolin-5-ones by cyclization of the oximes after acidification. This explains the existence of two GC peaks observed from urine extracts of patients under valproate therapy, which correspond to two tautomers of 2-isoxazolin-5-one originating from the oximes of the 3-keto-valproic acid.     

Unsaturated diether phospholipids in the Antartic methanogen Methanococcoides burtonii

Abstract The Antarctic methanogen Methanococcoides burtonii contained only diether phospholipids. These membrane components were analysed by gas chromatography and gas chromatography mass spectrometry. Of particular interest was the occurrence of unsaturated diether lipids in M. burtonii ; unsaturated ether lipids accounted for 57% of the diether phospholipids. To our knowledge, unsaturated ether lipids have not been previously reported in a methanogen. The presence of the unsaturated ether lipids in M. burtonii is probably the result of temperature adaptation by the bacterium. It may be possible to use these components as a chemical signature for methanogens in Antarctic and Southern Ocean environments.     

Isolation of 20 alpha and 20 beta 5 beta-pregnane-3 alpha,17 alpha,20,21-tetrol (hexahydro-Reichstein's compound-S) in a case of congenital adrenal hyperplasia

5β-Pregnane-3α, 17α, 20α, 21-tetrol (l) and 5β-pregnane-3α, 17α 20β, 21-tetrol (II) have been isolated and identified from the urine of a girl with congenital adrenal hyperplasia. The total 5β-pregnane-3α, 17α, 20(α+β),21-tetrol consisted of 60% of I and 40% of II. The final identity of the compounds was established by gas chromatography — mass spectrometry. The mass spectra of the two trimethylsilyl isomers were closely related to each other in contrast to the spectra of five other pairs of C₂₁-C-20(α and β)-hydroxy steroid-trimethylsilyl-ethers. The mass spectra of free I and II also exhibited many common features, but were less similar to each other than their trimethylsilyl derivatives.     

Microbiological conversion of 12-oxygenated and other derivatives of ent-kaur-16-en-19-oic acid by Gibberella Fujikuroi, mutant B1-41a

The metabolism of several ring C and D-functionalized ent-kaur-16-en-19-oic acids by cultures of Gibberella fujikuroi, mutant B1-41a, to the corresponding derivatives of the normal fungal gibberellins (GAs) and ent-kaurenoids is described. A range of 12α- and 12β-hydroxyGAs and ent-kaurenoids are characterized by their mass spectra and GC Kovats retention indices. The mass spectral and GC data are used to identify the 12α-hydroxy derivatives of GA₁₂, GA₁₄, GA₃₇ and GA₄ (GA₅₈), and of the 12β-hydroxy derivatives of ent-7α-hydroxy- and ent-6α, 7α-dihydroxykaurenoic acids, in seeds of Cucurbita maxima. Similarly the metabolites of GA₉, formed in seeds of Pisum sativum and cultures of G.fujikuroi, mutant B1-41a, are identified as 12α-hydroxyGA₉. ent-11β-Hydroxy- and ent-11-oxo-kaurenoic acids are metabolized by the fungus to the corresponding 11-oxygenated derivatives of the normal fungal ent-kaurenoids and some C₂₀-GAs; no 11-oxygenated C₁₉-GAs are formed. Grandiflorenic acid, 11β-hydroxygrandiflorenic acid, attractyligen and ent-15β-hydroxykaurenoic acid are metabolized to unidentified products.     

Fresh organically grown ginger (Zingiber officinale): composition and effects on LPS-induced PGE2 production

Gas chromatography in conjunction with mass spectrometry, a technique previously employed to analyze non-volatile pungent components of ginger extracts modified to trimethylsilyl derivatives, was applied successfully for the first time to analyze unmodified partially purified fractions from the dichloromethane extracts of organically grown samples of fresh Chinese white and Japanese yellow varieties of ginger, Zingiber officinale Roscoe (Zingiberaceae). This analysis resulted in the detection of 20 hitherto unknown natural products and 31 compounds previously reported as ginger constituents. These include paradols, dihydroparadols, gingerols, acetyl derivatives of gingerols, shogaols, 3-dihydroshogaols, gingerdiols, mono- and diacetyl derivatives of gingerdiols, 1-dehydrogingerdiones, diarylheptanoids, and methyl ether derivatives of some of these compounds. The thermal degradation of gingerols to gingerone, shogaols, and related compounds was demonstrated. The major constituent in the two varieties was [6]-gingerol, a chemical marker for Z. officinale. Mass spectral fragmentation patterns for all the compounds are described and interpreted. Anti-inflammatory activities of silica gel chromatography fractions were tested using an in vitro PGE2 assay. Most of the fractions containing gingerols and/or gingerol derivatives showed excellent inhibition of LPS-induced PGE2 production.     

Transformation of diphenyl ethers by Trametes versicolor and characterization of ring cleavage products

The white-rot fungi Trametes versicolor SBUG 1050, DSM 11269 and DSM 11309 are able to oxidize diphenyl ether and its halogenated derivatives 4-bromo- and 4-chlorodiphenyl ether. The products formed from diphenyl ether were 2- and 4-hydroxydiphenyl ether. Both 4-bromo- and 4-chlorodiphenyl ether were transformed to the corresponding products hydroxylated at the non-halogenated ring. Additionally, ring-cleavage products were detected by high perfomance liquid chromatography and characterized by gas chromatography/mass spectrometry and proton nuclear magnetic resonance spectroscopy. Unhalogenated diphenyl ether was degraded to 2-hydroxy-4-phenoxymuconic acid and 6-carboxy-4-phenoxy-2-pyrone. Brominated derivatives of both these compounds were formed from 4-bromodiphenyl ether, and 4-chlorodiphenyl ether was transformed in the same way to the analogous chlorinated ring cleavage products. Additionally, 4-bromo- and 4-chlorophenol were detected as intermediates from 4-bromo- and 4-chlorodiphenyl ether, respectively. In the presence of the cytochrome-P450 inhibitor 1-aminobenzotriazole, no metabolites were formed by cells of Trametes versicolor from the diphenyl ethers investigated. Cell-free supernatants of whole cultures with high laccase and manganese peroxidase activities were not able to transform the unhydroxylated diphenyl ethers used.     

Gas chromatographic-mass spectral characteristics of aporphine and tetrahydroprotoberberine alkaloids

The gas chromatographic (GC) and mass spectrometric (MS) properties of the silyl derivatives of aporphine and tetrahydroprotoberberine alkaloids are described. Selected representatives of these chemical classes of pharmacologically active bases were chromatographed on polar (OV-17) and nonpolar (OV-1) columns as their trimethylsilyl derivatives. The aporphines were eluted before the tetrahydroprotoberberines on both the polar and nonpolar columns. Simultaneous resolution of mixtures of aporphines and tetrahydroprotoberberines was readily achieved on the OV-1 column. An SE-30 column, used for combined GC-MS analysis, gave a similar resolution of these alkaloids. The mass spectra observed for the silylated 1,2,9,10-substituted aporphines were similar to those of underivatized aporphines, while the mass spectra of the silylated 1,2,10,11-substituted aporphines differed markedly from the spectra of the underivatized alkaloids. Although the mass spectra of the silylated derivatives of the 2,3,9,10- and 2,3,10,11-substituted tetrahydroprotoberberines were identical, these isomeric derivatives were separated by gas chromatography.     

Chromatographic behavior of oxygenated derivatives of cholesterol

Oxygenated derivatives of cholesterol have important functions in many biochemical processes. These oxysterols are difficult to study because of their low physiological concentrations, the facile formation of cholesterol autoxidation artifacts, and lack of information on their chromatographic behavior. Focusing on metabolites and autoxidation products of cholesterol, we have documented the chromatographic mobilities of 35 oxysterols under a variety of conditions: eight solvent systems for thin-layer chromatography on silica gel, several mobile phases for reversed-phase high-performance liquid chromatography (HPLC), and two types of stationary phase for capillary gas chromatography (GC) using trimethylsilyl derivatives. Notable differences in selectivity could be obtained by modifying the stationary or mobile phases. Separations of oxysterol pairs isomeric at side-chain carbons or C-7 were achieved on normal-phase, reversed-phase, chiral, or silver-ion HPLC columns. Chromatographic behavior is also described for side-chain hexadeuterated and heptafluorinated oxysterols, which are useful as standards in isotope dilution analyses and autoxidation studies, respectively. The overall results are relevant to many problems of oxysterol analysis, including the initial separation of oxysterols from cholesterol, determination of highly polar and nonpolar oxysterols, separation of isomeric pairs, selection of derivatization conditions for GC analysis, and quantitation of the extent of cholesterol autoxidation.     

Identification of polycyclic aromatic compounds in mutagenic emissions from steel casting

Workers in ferrous foundries show increased risk of lung cancer. In the steel casting process hot metal is poured into sand moulds solidified with organic binders, producing a plume of smoke containing a variety of organic compounds and showing strong mutagenicity in the Salmonella/S9 assay. We have collected the emissions produced when steel is poured into an experimental sand mould solidified with oil, clay and cereal, a widely used binder system. The organic constituents of these emissions have been fractionated by preparative reverse-phase high performance liquid chromatography (HPLC) and mutagenic fractions have been analysed by capillary column gas chromatography/mass spectrometry (GC/MS). Of the 65 compounds for which mass spectra are reported, 54 have been tentatively identified as alkyl derivatives of polycyclic aromatic compounds. Many compounds of this class are known to be carcinogenic and mutagenic. In addition, several unsubstituted polycyclic aromatic hydrocarbons, including the carcinogenic benz[a]anthracene and benzo[a]pyrene, were found to be present.     

Pentacyclic triterpenoids and sterols from seven species of mangrove

The isolation of pentacyclic triterpenoids from seven species of fresh mangrove leaves using a simple and rapid method is described. The leaves were homogenized using chloroform—methanol and the extract was diluted with water to precipitate out triterpenoids which were separated into neutral and acidic fractions. These were analysed by gas-liquid chromatography as acetyl and trimethylsilyl ether derivatives on a 3% OV-17 column. Sterols were isolated from the chloroform layer by preparative thin layer chromatography and were analysed by gas-liquid chromatography as their trimethylsilyl ether derivatives on a 3% OV-17 column. The triterpenoids found were α-amyrin, β-amyrin, lupeol, oleanolic acid and ursolic acid in most of the samples. Sterols found in all the samples were cholesterol, campesterol, stigmasterol, sitosterol and stigmast-7-en-3β-ol. Retention indices of the triterpenoids and sterols have been determined.