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T Ebner

Publications and source records attributed to T Ebner.

50 records · Page 3Linked to original sources
PubMed1993

Drug and xenobiotic glucuronidation catalysed by cloned human liver UDP-Glucuronosyltransferases stably expressed in tissue culture cell lines.

Two human UDP-Glucuronosyltransferase (UGT) cDNA clones were stably integrated into V79 chinese hamster fibroblast cells and the functional enzymes were expressed in this heterologous environment. More than 100 drugs and xenobiotics were used as substrates for glucuronidation, catalysed by the cloned UGTs to determine the chemical structures accepted as substrates. UGT HP1 exhibited a limited specificity for planar phenolic compounds, whereas UGT HP4 was more promiscuous in acceptance of non-planar phenols, anthraquinones, flavones, aliphatic alcohols, aromatic carboxylic acids, steroids and many drugs of varied structure. These conclusions are illustrated here by using a series of alkyl- and halophenols. This work indicates the considerable potential value in use of these recombinant cell lines to study human drug glucuronidation.

Animals↗
PubMed1993

Glucuronidation of thyroid hormone by human bilirubin and phenol UDP-glucuronyltransferase isoenzymes.

The glucuronidation of thyroid hormone by UDP-glucuronyltransferases (UGTs) stably transfected in Chinese hamster V79 lung fibroblasts was investigated. Human bilirubin UGT (HP3) and phenol UGT (HP4) both catalysed the glucuronidation of T4 and rT3, whereas glucuronidation of T3 was not significant, rT3 was the preferred substrate for both isoenzymes, glucuronidation rates being 1.6- and 6.4-times higher than conjugation of T4 by HP3 and HP4 clones, respectively. This is the first identification of thyroid hormone as potential alternative endogenous substrate for bilirubin UGT.

Animals↗
PubMed1993

The expression of UDP-glucuronosyltransferases of the UGT1 family in human liver and kidney and in response to drugs.

The expression of human UDP-glucuronosyltransferase (UGT) 1 gene family in the liver and kidney was examined using specific enzyme activity, antibodies and DNA probes for each of the four family members. Phenol UGT HP1 was expressed at a similar, relatively low, abundance in each liver and kidney whereas phenol UGT HP4 was more highly expressed in the kidney. Bilirubin UGTs (HP2 and HP3) were not detectable in the kidney and HP3 was the major isoform in the liver. The UGT activities towards certain specific substrates correlated well with the respective mRNA levels in the tissues. Bilirubin UGT HP3 was induced 2-3-fold in the livers from patients treated with phenytoin and phenobarbital. Storage of a human liver in University of Wisconsin solution which contains dexamethasone and insulin caused a large accumulation of all the UGT mRNAs, but these were not quantitatively translated into expressed UGT activities. The implications of these results are discussed.

Aged↗
PubMed1993

Multiple UDP-glucuronyltransferases for the glucuronidation of thyroid hormone with preference for 3,3',5'-triiodothyronine (reverse T3).

We have studied the glucuronidation of the thyroid hormones T4, T3 and rT3 by liver microsomes of Wistar, Gunn and WAG rats. Gunn rats have a defect in the gene coding for bilirubin and phenol UDP-glucuronyltransferase (UGT) isoenzymes; WAG rats have a genetic defect in androsterone UGT. In normal Wistar rats UGT activity was approximately 5-fold higher for rT3 than for T4 or T3. UGT activities for T4 and rT3, but not for T3, were impaired in Gunn rats. Conversely, UGT activity for T3, but not for T4 or rT3, was impaired in WAG rats. Thus, in rat liver rT3 is glucuronidated much more rapidly than T4 and T3. Our results support the view that T4 and rT3 are glucuronidated by bilirubin and phenol UGTs and T3 by androsterone UGT.

Androsterone↗
PubMed1993

The metabolism of aprindine in relation to the sparteine/debrisoquine polymorphism.

1. Incubation of the class I antiarrhythmic drug aprindine (AP) with human liver microsomes resulted in the formation of two hydroxylated metabolites (HA1 and HA2) and desethylaprindine which were identified by GC-mass spectrometry. In liver microsomes isolated from a poor metaboliser (PM) of sparteine no hydroxylated metabolites of AP were detected whereas AP N-dealkylation was unimpaired. Thus hydroxylation of AP is mediated by cytochrome P450 2D6 (CYP2D6). 2. AP was found to be a competitive inhibitor of CYP2D6 as indicated by its ability to impair the formation of (2S)-hydroxysparteine, 5,6-didehydrosparteine and 5-hydroxypropafenone by human liver microsomes. 3. These in vitro findings are consistent with a major role of CYP2D6 in the clearance of AP in vivo, with its ability to impair the metabolism of other CYP2D6 substrates in vivo, and an ability to cause phenocopying (conversion of extensive metaboliser phenotypes for sparteine/debrisoquine to apparent 'poor metabolisers).

Aprindine↗
PubMed1993

Human bilirubin UDP-glucuronosyltransferase catalyzes the glucuronidation of ethinylestradiol.

The synthetic estrogen ethinylestradiol is extensively eliminated as glucuronide metabolites in humans, but the UDP-glucuronosyltransferases (UGTs) catalyzing this reaction have not been identified. Therefore, ethinylestradiol was tested as a substrate for cloned human UGTs stably expressed in V79 cell lines. Two cloned expressed human enzymes, a bilirubin UGT and a phenol UGT, were observed to catalyze the glucuronidation of ethinylestradiol. High performance liquid chromatographic analysis of the products formed revealed that the expressed bilirubin UGT specifically produced ethinylestradiol-3-glucuronide. In human liver microsomes the ratio of 3-glucuronide/17-glucuronide was 97:3. Subsequent study of the cloned expressed enzymes and human liver microsomes from Crigler-Najjar patients by kinetic analysis and by substrate inhibition strongly indicated that a human liver bilirubin UGT was largely responsible for glucuronidation of ethinylestradiol. These results may provide an explanation for jaundice caused by ethinylestradiol in certain susceptible individuals.

Animals↗
PubMed1992

Characterisation of a human bilirubin UDP-glucuronosyltransferase stably expressed in hamster lung fibroblast cell cultures.

A cDNA encoding a human bilirubin UDP-glucuronosyltransferase has been isolated and stably expressed in Chinese hamster V79 lung fibroblast cell line. Western blotting of cell homogenates with anti-UGT antibody revealed a highly expressed protein of approx. 55.5 kDa in size. The expressed enzyme specifically catalysed the formation of bilirubin mono- and diglucuronides, and also catalysed the glucuronidation of two phenolic compounds, which are good substrates for other human UGT isoenzymes, at low rates.

Animals↗
PubMed1991

Cloning and stable expression of a new member of the human liver phenol/bilirubin: UDP-glucuronosyltransferase cDNA family.

A new human liver UDP-glucuronosyltransferase (HlugP4) has been cloned and expressed in cell culture. The expressed enzyme has a molecular mass of 56 kDa and preferentially catalysed the glucuronidation of halogenated and bulky alkyl phenols. The C-terminal half of the sequence (246 amino acids) is 96% identical with the same portion of HlugP1, whereas the N-terminal half of the deduced protein sequences are only 38% identical. These results suggest that the two isoenzymes may be derived from the same gene by differential splicing of the gene product.

Amino Acid Sequence↗
PubMed1991

Regioselectivity and stereoselectivity of the metabolism of the chiral quinolizidine alkaloids sparteine and pachycarpine in the rat.

1. The metabolism of (-)-sparteine and (+)-sparteine (pachycarpine) was investigated in male Sprague-Dawley rats by g.l.c.-mass spectrometry, and 13C- and 2H-n.m.r. spectroscopy. The structure of the major metabolite of (-)-sparteine was confirmed to be 2,3-didehydrosparteine by g.l.c.-mass spectrometry after alkaline sample work-up. 2H-n.m.r. spectroscopy showed that this metabolite exhibits the structure of the carbinolamine (2S)-hydroxysparteine in aqueous solution of neutral pH. No other metabolites with an enamine structure were observed by g.l.c.-mass spectrometry and 13C-n.m.r. spectroscopy. 2. Pachycarpine is metabolized in vivo and in vitro stereoselectively to the aliphatic alcohol (4S)-hydroxypachycarpine as the main metabolite. 3. The formation of the 2,3-didehydrosparteine proceeds via stereospecific abstraction of the axial 2 beta hydrogen atom. Inhibition in vitro studied with purified rat liver microsomes demonstrated that both sparteine enantiomers are metabolized by the same cytochrome P450 isozyme. Therefore this enzyme exhibits marked substrate and product stereoselectivity for the metabolism of the two enantiomeric quinolizidine alkaloids.

Administration, Oral↗
PubMed1989

[Stereospecific hydroxylation of (+)-sparteine (pachycarpine) in the rat].

Pachycarpine (4), the optical antipode of the lupine alkaloid (-)-sparteine (1), has been prepared from (-)-lupanine; its metabolism was studied in rats. After isolation and chromatographic purification, streochemically homogeneous (+)-(4S)-hydroxysparteine (7) was identified as the major urinary metabolite by use of mass spectrometry and high-field NMR-spectroscopy.

Animals↗
PubMedDate not supplied

Selection based on morphological assessment of oocytes and embryos at different stages of preimplantation development: a review.

In contrast to IVF, in ICSI the surrounding cumulus and corona cells must be removed completely, as only denuded oocytes can be successfully manipulated by the holding pipette. This ancillary effect of ICSI allows us to focus on the morphology of preimplantation development from the earliest stages. Early prognosis regarding the developmental fate of oocytes would help to limit a negative impact of culture conditions. However, little evidence is available that non-invasive selection at the oocyte stage (first polar body, granular cytoplasm) may be of prognostic value. Recently, certain patterns of pronuclei (number and the distribution of nucleoli) at the zygote stage were found to correlate with treatment outcome in IVF and ICSI cycles, offering an additional prognostic tool prior to cleavage. As there is evidence that embryo selection on day 2 or 3 based on morphological criteria (fragmentation, number of blastomeres, multinucleation, uneven cleavage) may be imprecise, patients might benefit from extended embryo culture to day 5. However, not all major chromosomal aberrations are incompatible with blastocyst formation, and prolonged culture in vitro does not exclusively select embryos with a normal chromosomal complement. Consequently, special care should be taken to minimize the presence of aneuploid concepti in culture. In addition, multiple selection at different stages of development will be required to filter out the correct 'candidate' embryo which will result in a healthy newborn.

Blastocyst↗
PubMedDate not supplied

A nuclear magnetic resonance study of sparteine delta metabolite structure.

Originally, the enamines 2,3- and 5,6-didehydrosparteine (2a and 3a, respectively) had been characterized by GC/MS as metabolites after administration of (-)-sparteine sulfate (1a.H2SO4). Since the existence of free enamines in aqueous medium seemed rather doubtful, the metabolism of (-)-sparteine was reinvestigated by high-resolution 1H-, 2H-, and 13C-NMR spectroscopy. When synthetic 1,2-didehydrosparteinium monoperchlorate (4a.ClO4) is dissolved in aqueous medium at pH 4-9, sterically uniform (2S)-hydroxysparteine (6a) is formed, as is proven unequivocally by 1H- and 13C-NMR. However, at pH less than or equal to 2, this carbinolamine eliminates water under reconstitution of the iminium structure 4a. No carbinolamine is formed, in contrast, from synthetic 1,6-didehydrosparteinium monoperchlorate (5a.ClO4). Upon oral application of [2R-2H]sparteine (1b), as the sulfate, the respective carbinolamine and iminium structures 6b and 5b are identified in the urines by 2H-NMR spectroscopy. Reversed-phase TLC likewise confirms the different structural principle of the two main sparteine metabolites. The free enamine bases, characterized by GC/MS, thus must be regarded as artifacts, formed in the work-up requisite for GC analysis.

Chromatography, Thin Layer↗
PubMedDate not supplied

Substrate specificities of two stably expressed human liver UDP-glucuronosyltransferases of the UGT1 gene family.

Two cloned human hepatic UDP-glucuronosyltransferase (UGT) cDNAs were stably expressed in chinese hamster V79 cells. More than 100 drugs and xenobiotics were used as substrates for glucuronidation catalyzed by the cloned human transferases to determine the chemical structures accepted as substrates. UGT HP1 exhibited a limited substrate specificity for planar phenolic compounds, whereas UGT HP4 was more promiscuous in acceptance of non-planar phenols, anthraquinones, flavones, aliphatic alcohols, aromatic carboxylic acids, steroids, and many drugs of varied structure. Levels of HP4 UGT activity toward some substrates were sufficient to allow determination of kinetic parameters for the enzyme reaction. Metabolism of drugs could be studied by addition to the recombinant cell lines in culture, and extraction of the media allowed analysis of glucuronide formation. Data presented herein demonstrate the potential of using these recombinant cell lines for investigation of phase II metabolism by human UGTs.

Animals↗