Detection and mass spectrometric characterization of novel long-term dehydrochloromethyltestosterone metabolites in human urine

The biotransformation of dehydrochloromethyltestosterone (DHCMT, 4-chloro-17β-hydroxy,17α-methylandrosta-1,4-dien-3-one) in man was studied with the aim to discover long-term metabolites valuable for the antidoping analysis. Having applied a high performance liquid chromatography for the fractionation of urinary extract obtained from the pool of several DHCMT positive urines, about 50 metabolites were found. Most of these metabolites were included in the GC-MS/MS screening method, which was subsequently applied to analyze the post-administration and routine doping control samples. As a result of this study, 6 new long-term metabolites were identified tentatively characterized using GC-MS and GC-MS/MS as 4-chloro-17α-methyl-5β-androstan-3α,16,17β-triol (M1), 4-chloro-18-nor-17β-hydroxymethyl,17α-methyl-5β-androsta-1,13-dien-3α-ol (M2), 4-chloro-18-nor-17β-hydroxymethyl,17α-methyl-5β-androst-13-en-3α-ol (M3), its epimer 4-chloro-18-nor-17α-hydroxymethyl,17β-methyl-5β-androst-13-en-3α-ol, 4-chloro-18-nor-17β-hydroxymethyl,17α-methylandrosta-4,13-dien-3α-ol (M4) and its epimer 4-chloro-18-nor-17α-hydroxymethyl,17β-methylandrosta-4,13-dien-3α-ol. The most long-term metabolite M3 was shown to be superior in the majority of cases to the other known DHCMT metabolites, such as 4-chloro-18-nor-17β-hydroxymethyl,17α-methylandrosta-1,4,13-trien-3-one and 4-chloro-3α,6β,17β-trihydroxy-17α-methyl-5β-androst-1-en-16-one.     

Incorporation of methionine-[methyl-2H3] and mevalonic acid-[2-14C,(4R)-4-3H1] into Phycomyces blakesleeanus sterols

Ergosterol, episterol, 4α-methyl-5α-ergosta-8,24(28)-dien-3β-ol and 24-methylene-24,25-dihydrolanosterol, isolated from Phycomyces blakesleeanus grown in the presence of methionine-[methyl-²H₃], each contained two deuterium atoms; lanosterol, however, was unlabelled. The ¹⁴C:³H atomic ratio of the following sterols isolated from P. blakesleeanus grown in the presence of mevalonic acid-[2-¹⁴C,(4R)-4-³H₁], was: ergosterol, 5:3; episterol, 5:4; ergosta-5,7,24(28)-trien-3β-ol, 5:3; 4α-methyl-5α-ergosta-8,24(28)-dien-3β-ol, 5:4; 24-methylene-24,25-dihydrolanosterol, 6:5; lanosterol, 6:5. The significance of these results in terms of ergosterol biosynthesis is discussed.     

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.     

The aromatization of (7 -3H) androstenedione by human placental mitochondria

A metabolite of 2,3-dihydroxyestra-1,3,5(10)-trien-17-one-6, 7-³H isolated from rat bile, was partially characterized by mass spectrometry as a methyl ether of 2,3,16-trihydroxyestra-1,3,5(10)-trien-17-one. The α configuration of the 16-hydroxy function was established by chromatographic comparison of the sodium borohydride reduced metabolite with synthetic 2-methoxy-estra-1, 3,5(10)-triene-3,16α,17β-triol and 2-methoxy-estra-1, 3,5(10)-triene-3,16β,17β-triol. The methyl group was located on the C-2 position by comparison with authentic 2- and 3- monomethyl ethers of 2,3-dihydroxy-estra-1, 3,5(10)-trien-17-one following pyrolytic removal of the 16α-hydroxyl group.3,16α-dihydroxy-2-methoxyestra-1,3,5(10)-trien-17-one was found to constitute 2% and 15% of the biliary radioactivity following administration of estrone-6,7-³H and 2,3-dihydroxyestra-1,3,5(10)-trien-17-one-6,7-³H respectively.     

Interactions between the active metabolite of tryptophan pyrolysate mutagen, N-hydroxy-Trp-P-2, and lipids: the role of lipid peroxides in the conversion of N-hydroxy-Trp-P-2 to non-reactive forms

The interactions between lipids and the mutagenic active metabolite of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-hydroxy-Trp-P-2), were studied. Oleic acid showed an inhibitory effect on the formation of this active metabolite mainly by inhibition of hepatic microsomal oxidation systems. On the other hand, microsomal lipids from rat liver and commercial pig liver lecithin diminished the amount of N-hydroxy-Trp-P-2 without inhibiting the metabolism of Trp-P-2. The direct reaction of these lipids with N-hydroxy-Trp-P-2 was disclosed by experiments using N-hydroxy-Trp-P-2 and lipids without microsomes. Furthermore, the participation of lipid peroxides in this reaction was suggested by a linear relationship between the concentrations of the conjugated diene of lipids and the disappearance of N-hydroxy-Trp-P-2. When [3H]N-hydroxy-Trp-P-2 was incubated in the presence of pig liver lecithin, the polar products which were not formed in the incubation without lipids were newly detected by thin-layer chromatography (TLC) analysis.     

Isolation of sterols from the marine fungus Corollospora lacera

Several marine fungi collected from the waters of Prince Edward Island, Canada, were screened for the presence of natural products exhibiting antibacterial activity. Both broths and mycelia of these fungi were studied using the bioassay-guided chromatographic separation. The 4 fractions from the extract of mycelia of Corollospora lacera exhibited weak antibacterial activity and were analyzed further. From these fractions, 2 sterols (5 alpha,8 alpha-epidioxyergosterol and 22E,24R-ergosta-7,22-diene-3beta,5 alpha,6 beta-triol) and a 3:1 mixture of linoleic and oleic acids were isolated. The presence of ergosterol was confirmed in dichloromethane extracts of mycelia of every fungus in this study and this sterol was isolated from the extract of mycelium of Corollospora lacera. Two other known compounds (5-hydroxymethylfuran-2-carbaldehyde and bis(2-ethylhexyl) phthalate), were isolated from the dichloromethane extract of mycelium of Monodictys pelagica. The phthalate was reported in the literature as a metabolite isolated from the fungi, but in our study it was proven to be an artifact of the culturing and (or) extraction procedures rather than a true fungal metabolite.     

Characterization of the microbial community in indoor environments: a chemical-analytical approach

An integrated procedure is presented whereby gas chromatography-ion trap mass spectrometry is used to determine chemical markers of gram-negative bacterial lipopolysaccharide (3-hydroxy fatty acids with 10 to 18 carbon atoms), gram-positive bacteria (branched-chain fatty acids with 15 and 17 carbon atoms), bacterial peptidoglycan (muramic acid), and fungal biomass (ergosterol) in samples of settled house dust. A hydrolysate of (13)C-labeled cyanobacterial cells is used as an internal standard for the first three markers. These analyses require two dust samples, one for 3-OH fatty acids, branched-chain fatty acids, and muramic acid and another for ergosterol. The method may be used to characterize microbial communities in environmental samples.     

Steroidal constituents of Solanum xanthocarpum

From the extract of the fruits of Solanum xanthocarpum (Solanaceae), five new steroidal compounds were isolated and characterized: 4α-methyl-24ξ-ethyl-5α-cholest-7-en-3β,22ξ-diol (1), 3β,22ξ-dihydroxy-4α-methyl-24ξ-ethyl-5α-cholest-7-en-6-one (2), 3β-benzoxy-14β,22ξ-dihydroxy-4α-methyl-24ξ-ethyl-5α-cholest-7-en-6-one (3), 3β-benzoxy-14α,22ξ-dihydroxy-4α-methyl-24ξ-ethyl-5α-cholest-7-en-6-one (4) and 3β-(p-hydroxy)-benzoxy-22ξ-hydroxy-4α-methyl-24ξ-ethyl-5α-cholest-7-en-6-one (5).     

Oxanthrone C-glycosides and epoxynaphthoquinol from the roots of Rumex japonicus

Five oxanthrone C-glycosides, namely rumejaposide A-E, and an epoxynaphthoquinol, together with eight known compounds, 2,6-dihydroxy benzoic acid, 4-hydroxy benzoic acid, epicatechin, 4-hydroxy-3-methoxy benzoic acid, 2,6-dimethoxy-4-hydroxyl benzoic acid, rutin, emodin and 2-acetyl-1,8-dihydroxy-3-methyl-6-methoxynaphthalene, were isolated from the roots of Rumex japonicus. The structures of the oxanthrone C-glycosides were elucidated by application of spectroscopic methods as (10R)10-C-beta-glucopyranosyl-1,8,10-trihydroxy-2-carboxyl-3-methyl-9(10H)-anthracenone, (10S)10-C-beta-glucopyranosyl-1,8,10-trihydroxy-2-carboxyl-3-methyl-9(10H)-anthracenone, (10R)10-C-beta-glucopyranosyl-1,6,8,10-tetrahydroxy-2-carboxyl-3-methyl-9(10H)-anthracenone, (10R)10-C-beta-glucopyranosyl-1,6,8,10-tetrahydroxy-3-hydroxymethyl-9(10H)-anthracenone, and (10R)10-C-beta-glucopyranosyl-1,6,8,10-tetrahydroxy-3-methyl-9(10H)-anthracenone. Absolute configurations for each compound were deduced by analyses of CD spectra and comparison with those known similar compounds. The structure of epoxynaphthoquinol was elucidated by spectroscopic methods as 3-acetyl-2-methyl-1,4,5-trihydroxy-2,3-epoxynaphthoquinol, and its relative configuration was determined by a 2D-ROESY experiment.     

Acetophenones and terpenoids from Senecio Gallicus

Six new compounds isolated from the aerial part of Senecio gallicus were: 7,11,15-trimethyl-3-methylene hexadecan-1,2-diol diacetate; 7,11,15-trimethyl-3-methylenehexadecan-1,2-diol; 3,5-bis(3-methyl-2-butenyl)-4-acetoxyacetophenone; 3-(2-hydroxy-3-methyl-3-butenyl)-5-(3-methyl-2-butenyl)4-hydroxacetophenone; 3-(2,3-dihydroxy-3-methyl-butyl)-5-(3-methyl-2-butenyl)-4-hydroxyacetophenone and 1,10-epoxy-8α-hydroxy eremophilenolide.     

Characterization of the Microbial Community in Indoor Environments: a Chemical-Analytical Approach

An integrated procedure is presented whereby gas chromatography-ion trap mass spectrometry is used to determine chemical markers of gram-negative bacterial lipopolysaccharide (3-hydroxy fatty acids with 10 to 18 carbon atoms), gram-positive bacteria (branched-chain fatty acids with 15 and 17 carbon atoms), bacterial peptidoglycan (muramic acid), and fungal biomass (ergosterol) in samples of settled house dust. A hydrolysate of ¹³C-labeled cyanobacterial cells is used as an internal standard for the first three markers. These analyses require two dust samples, one for 3-OH fatty acids, branched-chain fatty acids, and muramic acid and another for ergosterol. The method may be used to characterize microbial communities in environmental samples.     

Proton magnetic resonance spectra of 17ξ-Hydroxy-17ξ-methyl-5ξ-androstane C-3 ketone and c-3ξ alcohol isomers in chloroform-d and pyridine-d5

17α-Hydroxy-17β-methyl-5β-androstan-3-one, 17μ-methyl-5α-androstane-3α, 17α-diol, 17β-methyl-5α-androstane-3β, 17α-diol, 17α-methyl-5β-androstane-3β, 17β-diol, 17β-methyl-5β-androstane-3α, 17α-diol and 17β-methy1–5β-androstane-3β, 17α-diol were synthesized for the first time. ¹H NMR spectra of all four 17ξ-hydroxy/17ξ-methyl C-3 ketones and all eight C-3 alcohols were recorded in chloroform-d and pyridine-d₅. Pyridine-induced chemical shifts are discussed. Thin-layer Chromatographic data are given.     

Bicyclo[3,2,1]octanoid,neolignans from Aniba simulans

Six bicyclo[3,2,1]octanoid neolignans, isolated from the benzene extract of Aniba simulans Allen (Lauraceae) trunk wood, are shown to derive from two basic structures: 1-allyl-8-hydroxy-6-(3′-methoxy-4′,5′-methylenedioxyphenyl)-7-methyl-3-oxobicyclo[3,2,1]octane, substituted by 4-hydroxy, 4-hydroxy-5-methoxy, 4-methoxy or 4,5-dimethoxy groups; and 1-allyl-8-hydroxy-6-(3′-methoxy-4′,5′-methylenedioxyphenyl)-7-methyl-4-oxobicyclo[3,2,1]oct-2-ene, substituted by 3-hydroxy or 3-hydroxy-5-methoxy groups. The structural proposals are based on spectral data, interconversions synthesis of a derivative from the known (2R,3S,3aS)-3a-allyl-5-methoxy-2-(3′-methoxy,4′,5′-methylenedioxyphenyl)-3-methyl-2,3,3a,6-tetrahydro-6-oxobenzofuran.     

Indole alkaloids from two cultured cyanobacteria, Westiellopsis sp. and Fischerella muscicola

Chemical investigation of two cultured cyanobacteria, Westiellopsis sp. (SAG strain number 20.93) and Fischerella muscicola (UTEX strain number LB1829) led to the isolation of three hapalindole-type alkaloids, namely hapalindole X (1), deschloro hapalindole I (2), and 13-hydroxy dechlorofontonamide (3), along with ten known indole alkaloids (hapalindoles A, C, G, H, I, J, and U, hapalonamide H, anhydrohapaloxindole A, and fischerindole L) and fischerellins A and B. The structures were determined by a combination of spectroscopic analyses mainly based on 1D and 2D NMR and HRESIMS data. Selected compounds were evaluated for cytotoxicity and exhibited weak to moderate cytotoxicity against HT-29, MCF-7, NCI-H460, SF268, and IMR90 cells. All compounds, except hapalindole C, were evaluated for 20S proteasome inhibition and displayed either weak or no inhibition at 25μg/mL. Selected compounds were also evaluated for antimicrobial activity, and hapalindoles X (1) and A, and hapalonamide H showed potent activity against both Mycobacterium tuberculosis and Candida albicans with MIC values ranging from 0.6 to 2.5μM.     

Activation of a mutagen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole. Identification of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole and its reaction with DNA

A potent mutagen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), isolated from a tryptophan pyrolysate, was activated metabolically by rat liver microsomes and bound to DNA. An active metabolite formed by rat liver microsomes was identified as 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-OH-Trp-P-2). Synthetic N-OH-Trp-P-2 reacted with DNA efficiently after O-acetylation or to a lesser extent under acidic conditions (pH 5.5), but did not react appreciably under neutral conditions. Acid hydrolysis of DNA modified by O-acetylated N-OH-Trp-P-2 (N-OAc-Trp-P-2) gave 3-(8-guanyl)amino-1-methyl-5H-pyrido[4,3-b]indole (Gua-Trp-P-2), which is the main modified base of DNA formed by Trp-P-2 in the presence of microsomes. The glycoside bond of the modified base was found to be cleaved by heating at 100° for 1 hr at pH 7.0. In this way, the modified base was liberated from DNA modified by N-OAc-Trp-P-2 in good yield. N-OAc-Trp-P-2 bound to guanyl cytidine more effectively than to guanylic acid, suggesting that covalent binding with guanyl moiety of DNA involves intercalation of the ultimate mutagen into a base pair.     

The identification of 24-methylene-24,25-dihydrolanosterol and other possible ergosterol precursors in Phycomyces blakesleeanus and Agaricus campestris

The following sterols have been isolated from the fungi, Phycomyces blakesleeanus and Agaricus campestris: ergosterol, lanosterol, 24-methylene-24,25-dihydrolanosterol and episterol. 4,4-Dimethyl-5α-ergosta-8.24(28)-dien-3β-ol and 4α-methyl-5α-ergosta-8,24(28)-dien-3β-ol have been tentatively identified. Evidence for the incorporation of label from l-methionine-[methyl-¹⁴C] into some of these sterols in P. blakesleeanus has been obtained. The significance of these sterols in ergosterol biosynthesis is discussed.     

金顶侧耳次级代谢产物及抗氧化活性研究(英文)

采用硅胶柱色谱、ODS柱色谱、Sephadex LH-20柱色谱、HPLC等分离方法,对金顶侧耳大米发酵乙酸乙酯提取物进行分离;通过HR-ESI-MS、1H-NMR和13C-NMR等光谱学方法对其结构进行鉴定。并检测铁氰化钾还原能力、1,1-二苯基-2-苦基苯肼(DPPH)自由基清除能力和亚铁离子螯合能力。从金顶侧耳大米发酵粗提物中分离鉴定了吲哚甲醛、苯肽等6个化合物,吲哚甲醛为首次从该菌中分离得到,活性测试表明金顶侧耳粗提取物具有一定的还原能力,其大米发酵产物有可能开发成为一种新型功能性食品。     

Drechmerin H,a novel 1(2), 2(18)-diseco indole diterpenoid from the fungus Drechmeria sp. as a natural agonist of human pregnane X receptor

A novel 1(2), 2(18)-diseco indole diterpenoid, drechmerin H (1), was isolated from the fermentation broth of Drechmeria sp. together with a new indole diterpenoid, 2′-epi terpendole A (3), and a known analogue, terpendole A (2). Their structures were determined by HRESIMS, 1D and 2D NMR, ECD, and X-ray single crystal diffraction analyses as well as quantum chemical calculation. The abosulte configuration of terpendole A (2) was determined for the first time. Compound 1 displayed the significant agonistic effect on pregnane X receptor (PXR) with EC₅₀ value of 134.91 ± 2.01 nM, and its interaction with PXR was investigated by molecular docking. Meantime, a plausible biosynthetic pathway for compounds 1–3 is also discussed in the present work.     

A novel sterol from Chinese truffles Tuber indicum

From the fruiting bodies of Ascomycetes Tuber indicum, a new steroidal glucoside with polyhydroxy ergosterol nucleus, tuberoside (2), has been isolated along with additional four known ergosterol derivatives, (22E, 24R)-ergosta-7, 22-dien-3beta, 5alpha, 6beta-triol (1), 5alpha, 8alpha-epidioxy-(22E, 24R)-ergosta-6, 22-dien-3beta-ol (3), (22E, 24R)-ergosta-5, 22-dien-3beta-ol (4), and (22E, 24R)-ergosta-4, 6, 8(14), 22-tetraen-3-one (5). The structure of new compound was established as 3-O-beta-D-glucopyranosyl-(22E, 24R)-ergosta-7, 22-dien-5alpha, 6beta-diol (2) on the basis of chemical and spectroscopic means ((1)H NMR, (13)C NMR, HMQC, HMBC, MS, and IR). This is the first example of isolation of a polyhydroxylated ergosterol glucoside from higher fungi in nature.     

乳孔硫磺菌的化学成分和抗氧化活性

对乳孔硫磺菌子实体不同极性提取物进行了DPPH和ABTS自由基清除能力的测定,并对氯仿和乙酸乙酯提取物进行了分离纯化。结果表明,乳孔硫磺菌的石油醚、氯仿、乙酸乙酯和甲醇提取物均有一定的抗氧化活性,各提取物对两种自由基的清除能力均表现为甲醇提取物＞乙酸乙酯提取物＞氯仿提取物＞石油醚提取物,甲醇提取物对DPPH自由基的清除率最高可达到93.78%;对ABTS自由基的清除率最高可达到62.06%;从氯仿和乙酸乙酯提取物中分离得到10个化合物,分别是：(22E,24R)-5α,8α-过氧麦角甾-6,22-二烯-3β-醇（1）,阿里红酸 A（2）,麦角甾-7,22-二烯-3β-醇（3）,啤酒甾醇（4）,硫色多孔菌酸（5）,(4E,8E)-N-d-2′-hydroxypalmitoyl-1-O-β-d-glycopyranosyl- 9-methyl-4,8-sphingadienine（6）,麦角甾醇（7）,N-(2′-羟基二十四碳酰基)-1,3,4-三羟基-2-氨基-十八烷（8）,烟酸（9）和齿孔酸（10）。其中,化合物2、6、8和9为首次从硫磺菌属真菌中分离得到。