Biosynthesis of 16α,17α-epoxy-oestratrienol in rat liver microsomes

1980 ◽  
Vol 95 (1) ◽  
pp. 49-57 ◽  
Author(s):  
L. Siekmann ◽  
P. Thull ◽  
H. Breuer
Keyword(s):  
Reference Material ◽  
Thin Layer ◽  
Rat Liver ◽  
Mass Spectra ◽  
Styrene Oxide ◽  
Liver Microsomes ◽  
Mass Fragmentography ◽  
Rat Liver Microsomes ◽  
Epoxide Hydratase ◽  
Layer Chromatography

Abstract. After incubation of 1,3,5(10),16-oestratetraenol, a 16-dehydrosteroid, with rat liver microsomes, 16α,17α-epoxy-oestratrienol was isolated as metabolite. The compound was detected by the use of mass fragmentography after purification of the incubation extract by thin-layer chromatography. Since the epoxide is rapidly hydrolysed by a hepatic epoxide hydratase, only very small concentrations of this metabolite were present in the incubation extract. When styrene oxide was added to the incubation mixture as inhibitor of the epoxide hydratase, the yield of the steroid epoxide increased considerably. Final identification of the oestrogen epoxide was performed by recording mass spectra and by comparison with authentic reference material.

Biosynthesis of 16α,17α-epoxy-4-androsten-3-one in rat liver microsomes

1980 ◽  
Vol 95 (1) ◽  
pp. 58-66 ◽  
Author(s):  
B. Disse ◽  
L. Siekmann ◽  
H. Breuer
Keyword(s):  
Rat Liver ◽  
Styrene Oxide ◽  
Liver Microsomes ◽  
Epoxy Compound ◽  
Male Rat ◽  
Gas Liquid Chromatography ◽  
Rat Liver Microsomes ◽  
Epoxide Hydratase ◽  
Layer Chromatography ◽  
Generating System

Abstract. 4,16-Androstadien-3-one was incubated with the microsomal fraction of male rat liver in the presence of a NADPH generating system and oxygen. The metabolites formed were extracted from the incubation medium and purified by thin-layer chromatography (tic). Final identification was performed by combined gas liquid chromatography-mass spectrometry. Incubation of 4,16-androstadien-3-one resulted in the formation of a non-polar metabolite which proved to be 16α,17α-epoxy-4-androsten-3-one. This epoxide is a shortlived intermediate which is rapidly hydrolysed by the microsomal epoxide hydratase to 16β,17α-dihydroxy-4-androsten-3-one. In order to increase the amounts of epoxide in the incubation mixtures, styrene oxide which is a potent inhibitor of the epoxide hydratase was added. Under these conditions, up to 8% of the 16-dehydro-steroid incubated was transferred to the 16α,17α-epoxy-compound.

Metabolism of Coumarin and 4-Methylcoumarin by Rat-Liver Microsomes

1971 ◽  
Vol 49 (2) ◽  
pp. 177-184 ◽  
Author(s):  
P. A. Gibbs ◽  
K. Janakidevi ◽  
G. Feuer
Keyword(s):  
Fluorescence Spectroscopy ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
Rat Liver ◽  
Spectral Characteristics ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Hydroxyphenylacetic Acid ◽  
Layer Chromatography

The metabolism of coumarin and 4-methylcoumarin by rat-liver microsomes has been studied. The major metabolites of coumarin in vitro, viz. 3- and 7-hydroxycoumarin, o-hydroxyphenyllactic acid, and o-hydroxyphenylacetic acid, were identified by thin-layer chromatography, and ultraviolet and fluorescence spectroscopy. Metabolites derived from 4-methylcoumarin in vitro have shown similar chromatographic and spectral characteristics as those of coumarin, suggesting that they are the methyl analogues of corresponding coumarin metabolites.

Use of recombinant human ferrochelatase as a sensitive bioassay for N-alkylprotoporphyrin IX formed after interaction of porphyrinogenic xenobiotics with rat liver microsomes

2000 ◽  
Vol 78 (7) ◽  
pp. 578-581 ◽  
Author(s):  
Jeremy T Gamble ◽  
Simon GW Wong ◽  
Harry A Dailey ◽  
Gerald S Marks
Keyword(s):  
Cytochrome P450 ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Bioassay System ◽  
Human Cytochrome ◽  
In Vitro Systems ◽  
Layer Chromatography ◽  
Cyp Isozymes

Several porphyrinogenic xenobiotics elicit mechanism-based inactivation of cytochrome P450 (CYP) isozymes, leading to the formation of N-alkylprotoporphyrin IX (N-alkylPP), a potent inhibitor of ferrochelatase, the terminal enzyme in heme biosynthesis. Recognizing their role in experimental porphyria, our long term objective is the establishment of an appropriate in vitro system for the detection and quantification of N-alkylPPs, formed in human liver after the administration of potential porphyrinogenic compounds. In a previous study, we used a combination of thin-layer chromatography and UV-visible spectrophotometry to isolate and identify N-alkylPPs after incubating porphyrinogenic compounds with rat liver microsomes. However, the overall yield of N-alkylPPs was low, and it was concluded that in vitro systems, such as human lymphoblastoid microsomal preparations containing single cDNA-expressed human cytochrome P450 (CYP) isozymes, do not contain sufficient CYP for in vitro studies designed to isolate N-alkylPP. In the present study we demonstrate that purified recombinant human ferrochelatase (FC) provides an extremely sensitive bioassay system for N-alkylPPs and is capable of detecting N-alkylPP in the 10-6nmol range. Therefore, we propose that this bioassay system might allow the use of human lymphoblastoid microsomal preparations containing single cDNA-expressed human CYP isozymes to detect N-alkylPP produced after mechanism-based (catalysis-based) CYP inactivation. If this is found to be correct it will facilitate identification of potentially porphyrinogenic drugs prior to administration to humans.Key words: ferrochelatase, N-alkylprotoporphyrin IX, porphyria, mechanism-based inactivation.

STUDIES ON THE METABOLISM OF C19 STEROIDS IN RAT LIVER. 6. HYDROXYLATION OF 4-ANDROSTENE-3,17-DIONE IN RAT LIVER MICROSOMES

1970 ◽  
Vol 65 (1) ◽  
pp. 84-94 ◽  
Author(s):  
Jan-Åke Gustafsson ◽  
Belisário P. Lisboa
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Gas Chromatography Mass Spectrometry ◽  
Male Rat ◽  
Rat Liver Microsomes ◽  
Relative Importance ◽  
Adult Male Rat ◽  
Layer Chromatography

ABSTRACT Following incubations of androstenedione with 105 000 × g microsomes of adult male rat liver, 6β-, 6α-, 7α-, 16α-, and 18-hydroxyandrostenedione were isolated by thin-layer chromatography and identified by gas chromatography-mass spectrometry. After incubations with testosterone the only 3,17-dioxo-Δ4-steroids formed were 6β- and 7α-hydroxyandrostenedione. 6β- and 18-Hydroxyandrostenedione were isolated after incubations with 6β- and 18-hydroxytestosterone, respectively. The relative importance of the 17-oxo- and the 17β-hydroxy-pathways in the formation of 3,17-dioxo-Δ4-C19O3 steroids is discussed.

Metabolic Analysis of Triptolide Microspheres in Human, Dog, Rabbit and Rat Liver Microsomes with UPLC-MS/MS Method

2020 ◽  
Vol 17 ◽  
Author(s):  
LiJuan Wang ◽  
Yan Liu ◽  
Rui Li ◽  
DongXian He
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Internal Standard ◽  
Gradient Elution ◽  
Rat Liver Microsomes ◽  
Good Precision ◽  
Kinetics Parameters ◽  
Measure Concentration ◽  
Standard Curve ◽  
Variation Of Parameters

Objectives: Triptolide (TPL) has been shown to have a good clinical effect on rheumatoid arthritis (RA). We designed TPL microspheres (TPL-MS) and investigated its metabolic behavior in human, dog, rabbit and rat liver microsomes (HLM, DLM, RLM and SDRLM) with UPLC-MS/MS method. Methods: First, a UPLC-MS/MS method was established to measure concentration of TPL in samples. The sample was separated on a C18 column (2.1×100 mm, 1.8μm) and eluted with a gradient elution. The precursor ion/product ion were m/z 378.1/361.0 for TPL and 260.0/116.2 for the internal standard. Then T1/2, Vmax and CLint were calculated from the above data. Finally, the metabolites of TPL-MS were identified by high-resolution UPLC-MS/MS. The sample was separated on a C18 column (2.1×100 mm, 2.2 μm) and eluted with isocratic elution. Mass spectrometric detection was carried out on a thermo Q-exactive mass spectrometer with HESI. The scanning range of precursor ions was from m/z 50 to m/z 750. Result and Discussion: Through several indicators including standard curve, precision, accuracy, stability, matrix effect and recovery rate, the enzymatic kinetics parameters including T1/2, Vmax and CLint were completed. Several metabolites of TPL-MS were identified. Conclusion: UPLC-MS/MS method is an accurate and sensitive method for determination of TPL in liver microsome samples with good precision, accuracy and stability. The variation of parameters indicated that the microspheres can delay the elimination of TPL in liver microsomes. The metabolism of TPL-MS varied among species, but no new metabolites appeared.

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