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Asbjorg S Christophersen - One of the best experts on this subject based on the ideXlab platform.
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Effects of Ethanol on Ethylmorphine Metabolism in Isolated Rat Hepatocytes: Characterization by Means of a Multicompartmental Model
Pharmacology & Toxicology, 1997Co-Authors: Bang Qian Xu, Tor A. Aasmundstad, Bjern Lillekjendlie, Anders Bjørneboe, Asbjorg S Christophersen, Jorg MorlandAbstract:Hepatic cytochrome P-450 enzymes mediate at least two important biotransformation pathways of codeine and Ethylmorphine starting with either N-demethylation or 0-dealkylation, producing polar metabolites which are then subsequently glucuronidated. The present study was designed to characterise the acute effects of ethanol on the metab- olism of Ethylmorphine and to compare it with the effects on codeine in suspensions of freshly isolated rat hepatocytes. Isolated rat hepatocytes from male Wistar rats were prepared by a collagenase perfusion method. Ethylmorphine, codeine and their metabolites were quantified by HPLC with UV detection. The total Ethylmorphine elimination rate was reduced by 12% at 5 mM and 38% at 100 mM ethanol. The corresponding percentages for codeine were 16 and 43%. In the presence of ethanol the concentrations of several intermediate and end products of Ethylmorphine and codeine changed markedly from the control situation. The experimental data were applied to a mathematical compartmental linear model to estimate the influence of ethanol on the separate reaction rates in the two main metabolic pathways. The ratios between reaction rate constants in the Ethylmorphine experiments at 100 and 0 mM ethanol were 0.65 for Ethylmorphine-tnorethyl- morphine, 0.63 for norEthylmorphine-tnormorphine, 0.56 for Ethylmorphine-tmorphine, 0.49 for morphine+normor- phine, 0.31 for normorphine-tnormorphine-3-glucuronide and 0.49 for morphine-tmorphine-3-glucuronide. similar effects of ethanol on codeine metabolism were found. In additional experiments, norEthylmorphine or norcodeine (50 pM) was incubated with 5 mM to 100 mM of ethanol and the metabolism of both norEthylmorphine and norcodeine was found to be inhibited by ethanol in a concentration-dependent manner. The glucuronidation of morphine and nor- morphine added in separate experiments was also inhibited by ethanol, from 22 to 36% for morphine-3-glucuronide and 30 to 60% for normorphine-3-glucuronide, respectively, in the presence of 5 mM to 100 mM of ethanol. It was concluded that all steps in the metabolism of Ethylmorphine (and codeine) leading to the end products morphine-3-glucuronide and normorphine-3-glucuronide were inhibited by ethanol, and that the glucuronidation processes were the ones most affected by ethanol.
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biotransformation and pharmacokinetics of Ethylmorphine after a single oral dose
British Journal of Clinical Pharmacology, 1995Co-Authors: Tor A. Aasmundstad, Anders Bjørneboe, Asbjorg S Christophersen, Bq Q Xu, I Johansson, A Ripel, E Bodd, Jorg MorlandAbstract:: 1. The pharmacokinetics of Ethylmorphine after administration of a single dose of the cough mixture Cosylan were investigated in 10 healthy subjects. 2. The median urinary recovery of Ethylmorphine and measured metabolites was 77% over 48 h. The median tmax of unchanged Ethylmorphine was 45 min, and the terminal elimination t1/2 was 2 h. Ethylmorphine-6-glucuronide was found to be the major metabolite. 3. Two subjects had significantly lower urinary recovery (0.48 h) of morphine and morphine-glucuronides than the remainder. Furthermore, these two had urinary metabolic ratios (MRO) and partial metabolic clearances (CLmO) for O-deethylation of Ethylmorphine tentatively classifying them phenotypically as poor metabolisers of the debrisoquine/sparteine type. 4. Genotyping for cytochrome P450 (CYP) 2D6 alleles revealed five homozygote (wt/wt) and five heterozygote subjects. Two subjects phenotypically classified as poor metabolisers were genotypically CYP2D6A/wt and CYP2D6D/wt, respectively. 5. Serum and urine samples taken more than 8 and 24 h after administration of ethyl-morphine respectively, contained morphine and morphine-glucuronides, but no Ethylmorphine, Ethylmorphine-6-glucuronide or (serum only) norEthylmorphine. NorEthylmorphine could be detected after hydrolysis of urine samples in all subjects. The urinary recovery of the active metabolites morphine and morphine-6-glucuronide after administration of Ethylmorphine varied by a factor of 9 between individuals. 6. The wide variation in recovery of morphine and morphine-glucuronides after oral administration of Ethylmorphine could not be explained simply by a difference in CYP2D6 genotype. Constitutional variation in other enzymatic pathways involved in Ethylmorphine metabolism is probably crucial. Ratios of morphine to parent drug cannot be used to distinguish the source of morphine after administration of Ethylmorphine. NorEthylmorphine should be included in urine assays for opiates in forensic toxicology, and no firm conclusions about the source of morphine are possible based on serum samples obtained more than 24 h after drug administration.
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Ethylmorphine metabolism in isolated rat hepatocytes
Pharmacology & Toxicology, 1993Co-Authors: Bang Qian Xu, Tor A. Aasmundstad, Anders Bjørneboe, Asbjorg S Christophersen, Ase Ripel, Jorg MorlandAbstract:: The metabolism of Ethylmorphine has been studied in suspensions of isolated rat hepatocytes. Early during incubation, the two major metabolic intermediates detected were morphine and norEthylmorphine following N- and O-dealkylation of Ethylmorphine, respectively. During subsequent incubation the concentration of the second metabolic intermediate, normorphine increased, before the concentration peaked at approximately 20 microM (100 microM Ethylmorphine). Both morphine and normorphine were glucuronidated to form morphine-3-glucuronide and normorphine-3-glucuronide, respectively, which appeared to be the major metabolic end products. The percentage of Ethylmorphine metabolized to morphine-3-glucuronide was found to be dependent on the initial concentration of Ethylmorphine. With increasing initial Ethylmorphine concentration the relative formation of morphine-3-glucuronide was reduced (29 +/- 10% at 5 microM, 18 +/- 5% at 20 microM, and 15 +/- 4% at 100 microM mean +/- S.D., n = 10). The concentrations of Ethylmorphine and its metabolites were found to be higher in liver cells than in medium. Thus the ratios between the intra-/extra-cellular concentrations of Ethylmorphine increased somewhat from an initial value of 4 during the period for which Ethylmorphine could be detected intracellularly. The drug metabolites all exhibited ratios above 10 for the initial 100 min. of incubation. With time these ratios showed a decline, but even for prolonged incubation the ratios were 5 or higher for the end products. Thus considerable drug concentration gradients existed across the cell membrane of isolated rat hepatocytes.
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evaluation of a method for simultaneous quantification of codeine Ethylmorphine and morphine in blood
Forensic Science International, 1991Co-Authors: Hallvard Gjerde, Unni Fongen, Hans Gundersen, Asbjorg S ChristophersenAbstract:Abstract Codeine, Ethylmorphine and morphine are the most commonly detected opiates in forensic blood samples in Norway. A method for the simultaneous quantification of these opiates utilizing solid phase extraction and gas chromatography-mass spectrometry has been evaluated. The detection limits were 0.026 μmol/l for codeine, 0.025 μmol/l for Ethylmorphine and 0.032 μmol/l for morphine (corresponding to 7.8, 7.8 and 9.1 μg/l, respectively). The analytical variations at concentrations of 1.0 μmol/l codeine, 1.0 μmol/l Ethylmorphine and 0.5 μmol/l morphine were less than 5%.
Jorg Morland - One of the best experts on this subject based on the ideXlab platform.
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Effects of Ethanol on Ethylmorphine Metabolism in Isolated Rat Hepatocytes: Characterization by Means of a Multicompartmental Model
Pharmacology & Toxicology, 1997Co-Authors: Bang Qian Xu, Tor A. Aasmundstad, Bjern Lillekjendlie, Anders Bjørneboe, Asbjorg S Christophersen, Jorg MorlandAbstract:Hepatic cytochrome P-450 enzymes mediate at least two important biotransformation pathways of codeine and Ethylmorphine starting with either N-demethylation or 0-dealkylation, producing polar metabolites which are then subsequently glucuronidated. The present study was designed to characterise the acute effects of ethanol on the metab- olism of Ethylmorphine and to compare it with the effects on codeine in suspensions of freshly isolated rat hepatocytes. Isolated rat hepatocytes from male Wistar rats were prepared by a collagenase perfusion method. Ethylmorphine, codeine and their metabolites were quantified by HPLC with UV detection. The total Ethylmorphine elimination rate was reduced by 12% at 5 mM and 38% at 100 mM ethanol. The corresponding percentages for codeine were 16 and 43%. In the presence of ethanol the concentrations of several intermediate and end products of Ethylmorphine and codeine changed markedly from the control situation. The experimental data were applied to a mathematical compartmental linear model to estimate the influence of ethanol on the separate reaction rates in the two main metabolic pathways. The ratios between reaction rate constants in the Ethylmorphine experiments at 100 and 0 mM ethanol were 0.65 for Ethylmorphine-tnorethyl- morphine, 0.63 for norEthylmorphine-tnormorphine, 0.56 for Ethylmorphine-tmorphine, 0.49 for morphine+normor- phine, 0.31 for normorphine-tnormorphine-3-glucuronide and 0.49 for morphine-tmorphine-3-glucuronide. similar effects of ethanol on codeine metabolism were found. In additional experiments, norEthylmorphine or norcodeine (50 pM) was incubated with 5 mM to 100 mM of ethanol and the metabolism of both norEthylmorphine and norcodeine was found to be inhibited by ethanol in a concentration-dependent manner. The glucuronidation of morphine and nor- morphine added in separate experiments was also inhibited by ethanol, from 22 to 36% for morphine-3-glucuronide and 30 to 60% for normorphine-3-glucuronide, respectively, in the presence of 5 mM to 100 mM of ethanol. It was concluded that all steps in the metabolism of Ethylmorphine (and codeine) leading to the end products morphine-3-glucuronide and normorphine-3-glucuronide were inhibited by ethanol, and that the glucuronidation processes were the ones most affected by ethanol.
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biotransformation and pharmacokinetics of Ethylmorphine after a single oral dose
British Journal of Clinical Pharmacology, 1995Co-Authors: Tor A. Aasmundstad, Anders Bjørneboe, Asbjorg S Christophersen, Bq Q Xu, I Johansson, A Ripel, E Bodd, Jorg MorlandAbstract:: 1. The pharmacokinetics of Ethylmorphine after administration of a single dose of the cough mixture Cosylan were investigated in 10 healthy subjects. 2. The median urinary recovery of Ethylmorphine and measured metabolites was 77% over 48 h. The median tmax of unchanged Ethylmorphine was 45 min, and the terminal elimination t1/2 was 2 h. Ethylmorphine-6-glucuronide was found to be the major metabolite. 3. Two subjects had significantly lower urinary recovery (0.48 h) of morphine and morphine-glucuronides than the remainder. Furthermore, these two had urinary metabolic ratios (MRO) and partial metabolic clearances (CLmO) for O-deethylation of Ethylmorphine tentatively classifying them phenotypically as poor metabolisers of the debrisoquine/sparteine type. 4. Genotyping for cytochrome P450 (CYP) 2D6 alleles revealed five homozygote (wt/wt) and five heterozygote subjects. Two subjects phenotypically classified as poor metabolisers were genotypically CYP2D6A/wt and CYP2D6D/wt, respectively. 5. Serum and urine samples taken more than 8 and 24 h after administration of ethyl-morphine respectively, contained morphine and morphine-glucuronides, but no Ethylmorphine, Ethylmorphine-6-glucuronide or (serum only) norEthylmorphine. NorEthylmorphine could be detected after hydrolysis of urine samples in all subjects. The urinary recovery of the active metabolites morphine and morphine-6-glucuronide after administration of Ethylmorphine varied by a factor of 9 between individuals. 6. The wide variation in recovery of morphine and morphine-glucuronides after oral administration of Ethylmorphine could not be explained simply by a difference in CYP2D6 genotype. Constitutional variation in other enzymatic pathways involved in Ethylmorphine metabolism is probably crucial. Ratios of morphine to parent drug cannot be used to distinguish the source of morphine after administration of Ethylmorphine. NorEthylmorphine should be included in urine assays for opiates in forensic toxicology, and no firm conclusions about the source of morphine are possible based on serum samples obtained more than 24 h after drug administration.
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Ethylmorphine metabolism in isolated rat hepatocytes
Pharmacology & Toxicology, 1993Co-Authors: Bang Qian Xu, Tor A. Aasmundstad, Anders Bjørneboe, Asbjorg S Christophersen, Ase Ripel, Jorg MorlandAbstract:: The metabolism of Ethylmorphine has been studied in suspensions of isolated rat hepatocytes. Early during incubation, the two major metabolic intermediates detected were morphine and norEthylmorphine following N- and O-dealkylation of Ethylmorphine, respectively. During subsequent incubation the concentration of the second metabolic intermediate, normorphine increased, before the concentration peaked at approximately 20 microM (100 microM Ethylmorphine). Both morphine and normorphine were glucuronidated to form morphine-3-glucuronide and normorphine-3-glucuronide, respectively, which appeared to be the major metabolic end products. The percentage of Ethylmorphine metabolized to morphine-3-glucuronide was found to be dependent on the initial concentration of Ethylmorphine. With increasing initial Ethylmorphine concentration the relative formation of morphine-3-glucuronide was reduced (29 +/- 10% at 5 microM, 18 +/- 5% at 20 microM, and 15 +/- 4% at 100 microM mean +/- S.D., n = 10). The concentrations of Ethylmorphine and its metabolites were found to be higher in liver cells than in medium. Thus the ratios between the intra-/extra-cellular concentrations of Ethylmorphine increased somewhat from an initial value of 4 during the period for which Ethylmorphine could be detected intracellularly. The drug metabolites all exhibited ratios above 10 for the initial 100 min. of incubation. With time these ratios showed a decline, but even for prolonged incubation the ratios were 5 or higher for the end products. Thus considerable drug concentration gradients existed across the cell membrane of isolated rat hepatocytes.
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A case of high opiate tolerance: implications for drug analyses and interpretations.
International Journal of Legal Medicine, 1991Co-Authors: Hallvard Gjerde, Jorg MorlandAbstract:A case of driving under the influence of extremely high concentrations of codeine and Ethylmorphine is reported. A high blood concentration of morphine was also found, which in this case was probably a metabolic product of codeine and Ethylmorphine. This illustrates that when morphine is found in blood, the sample should also be analysed for other opiates in order to avoid misinterpretations. Language: en
Anders Rane - One of the best experts on this subject based on the ideXlab platform.
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evidence for a role of cytochrome p450 2d6 and 3a4 in Ethylmorphine metabolism
British Journal of Clinical Pharmacology, 1995Co-Authors: O Mortimer, C A D Smith, C R Wolf, Anders RaneAbstract:Ethylmorphine is metabolised by N-demethylation (to norEthylmorphine) and by O-deethylation (to morphine). The O-deethylation reaction was previously shown in vivo to co-segregate with the O-demethylation of dextromethorphan indicating that Ethylmorphine is a substrate of polymorphic cytochrome P450(CYP)2D6. To study further the features of Ethylmorphine metabolism we investigated its N-demethylation and O-deethylation in human liver microsomes from eight extensive (EM) and one poor metaboliser (PM) of dextromethorphan. Whereas N-demethylation varied only two-fold there was a 4.3-fold variation in the O-deethylation of Ethylmorphine, the lowest rate being observed in the PM. Quinidine, at a concentration of 1 microM, inhibited O-deethylation in microsomes from an EM, but was unable to do so in microsomes from the PM. The immunoidentified CYP2D6 and CYP3A4 correlated with the rates of O-deethylation (r = 0.972) and N-demethylation (r = 0.969), respectively. We conclude that the O-deethylation of Ethylmorphine is catalysed by the CYP2D6 in human liver microsomes consistent with previous findings in healthy volunteers.
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comparison of human fetal hepatic and adrenal cytochrome p450 activities with some major gestational steroids and Ethylmorphine as substrates
The Journal of Steroid Biochemistry and Molecular Biology, 1992Co-Authors: Anders Rane, Stig Henningsson, Margarita G LadonaAbstract:Abstract The immunoidentified human fetal liver and adrenal microsomal contents of cytochromes P450IIIA and P450VIIA1 were compared to the metabolism of steroids and Ethylmorphine. In fetal liver microsomes, 16α-hydroxylation of dehydroepiandrosterone (DHA) was catalyzed at a high rate in almost all investigated specimens and accompanied by a high Ethylmorphine N-demethylase activity. Progesterone 16α- and 17α-hydroxylation was found only in the livers with the highest DHA 16α-hydroxylation activities, while 21-hydroxylation of progesterone was catalyzed only occasionally in these samples. In fetal adrenal microsomes, 21-hydroxylation of progesterone to 11-desoxycorticosterone (DOC) and 11-desoxycortisol (DOCOL) was catalyzed. In contrast to fetal liver, the adrenals also catalyzed the 17α-hydroxylation of pregnenolone and the formation of DHA from 17α-OH-pregnenolone. 16α-hydroxylation of DHA and Ethylmorphine N-demethylation were modest in the adrenals. P450IIIA/HLp was immunoidentified in all investigated liver specimens except two ( 18 20 ) in which no Ethylmorphine N-demethylation or 16α-hydroxylation of DHA was found. P450XVIIA1 bands were observed in 8 20 blots of liver specimens, but there was no correlation between the density of these bands and the 17α-hydroxylation of progesterone. All 11 fetal adrenal samples catalyzed DHA 16α-hydroxylation, although only 8 were positive for P450IIIA/HLp. All investigated adrenals were positive in regard of the P450XVIIA1 band, except one ( 8 9 ) with a low 17α-hydroxylation of progesterone. All adrenal specimens catalyzed 21-hydroxylation of progesterone and contained P450C21 bands in immunoblots and all samples catalyzed the formation of DOC and DOCOL from progesterone. Our findings in the fetal livers show a correlation between the DHA 16α-hydroxylation and immunoidentified P450IIIA/HLp bands. In adrenals, there was a correlation between the immunoidentified P450XVIIIA1 bands and the 17α-hydroxylation of progesterone.
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Ethylmorphine o deethylation cosegregates with the debrisoquin genetic metabolic polymorphism
Clinical Pharmacology & Therapeutics, 1992Co-Authors: Anders Rane, Ali R Modiri, Eva GerdinAbstract:The single oral dose kinetics of Ethylmorphine and its fractional metabolic clearance by O-dealkylation and N-dealkylation was investigated in five extensive and four poor metabolizers of dextromethorphan. In addition, the urinary metabolic ratios for these pathways (MRO and MRN, respectively) were investigated in a larger group of 27 extensive metabolizers and six poor metabolizers. The mean values for the fractional metabolic clearance by O-dealkylation and the MRO differed significantly between the poor metabolizers and extensive metabolizers without overlap between the values of either of these parameters in the two groups of subjects. In contrast, the corresponding parameters for the N-demethylation did not differ between poor metabolizers and extensive metabolizers. The area under the plasma concentration versus time curve was significantly higher (about three times higher) in the poor metabolizers compared with the extensive metabolizers (p = 0.004). Our data suggest that Ethylmorphine is O-deethylated by the cytochrome P4502D6 isozyme inasmuch as both the fractional metabolic clearance by O-dealkylation and the MRO were found to cosegregate with the phenotype for the O-demethylation of dextromethorphan in our group of subjects. Clinical Pharmacology and Therapeutics (1992) 52, 257–264; doi:10.1038/clpt.1992.139
Tor A. Aasmundstad - One of the best experts on this subject based on the ideXlab platform.
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Effects of Ethanol on Ethylmorphine Metabolism in Isolated Rat Hepatocytes: Characterization by Means of a Multicompartmental Model
Pharmacology & Toxicology, 1997Co-Authors: Bang Qian Xu, Tor A. Aasmundstad, Bjern Lillekjendlie, Anders Bjørneboe, Asbjorg S Christophersen, Jorg MorlandAbstract:Hepatic cytochrome P-450 enzymes mediate at least two important biotransformation pathways of codeine and Ethylmorphine starting with either N-demethylation or 0-dealkylation, producing polar metabolites which are then subsequently glucuronidated. The present study was designed to characterise the acute effects of ethanol on the metab- olism of Ethylmorphine and to compare it with the effects on codeine in suspensions of freshly isolated rat hepatocytes. Isolated rat hepatocytes from male Wistar rats were prepared by a collagenase perfusion method. Ethylmorphine, codeine and their metabolites were quantified by HPLC with UV detection. The total Ethylmorphine elimination rate was reduced by 12% at 5 mM and 38% at 100 mM ethanol. The corresponding percentages for codeine were 16 and 43%. In the presence of ethanol the concentrations of several intermediate and end products of Ethylmorphine and codeine changed markedly from the control situation. The experimental data were applied to a mathematical compartmental linear model to estimate the influence of ethanol on the separate reaction rates in the two main metabolic pathways. The ratios between reaction rate constants in the Ethylmorphine experiments at 100 and 0 mM ethanol were 0.65 for Ethylmorphine-tnorethyl- morphine, 0.63 for norEthylmorphine-tnormorphine, 0.56 for Ethylmorphine-tmorphine, 0.49 for morphine+normor- phine, 0.31 for normorphine-tnormorphine-3-glucuronide and 0.49 for morphine-tmorphine-3-glucuronide. similar effects of ethanol on codeine metabolism were found. In additional experiments, norEthylmorphine or norcodeine (50 pM) was incubated with 5 mM to 100 mM of ethanol and the metabolism of both norEthylmorphine and norcodeine was found to be inhibited by ethanol in a concentration-dependent manner. The glucuronidation of morphine and nor- morphine added in separate experiments was also inhibited by ethanol, from 22 to 36% for morphine-3-glucuronide and 30 to 60% for normorphine-3-glucuronide, respectively, in the presence of 5 mM to 100 mM of ethanol. It was concluded that all steps in the metabolism of Ethylmorphine (and codeine) leading to the end products morphine-3-glucuronide and normorphine-3-glucuronide were inhibited by ethanol, and that the glucuronidation processes were the ones most affected by ethanol.
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biotransformation and pharmacokinetics of Ethylmorphine after a single oral dose
British Journal of Clinical Pharmacology, 1995Co-Authors: Tor A. Aasmundstad, Anders Bjørneboe, Asbjorg S Christophersen, Bq Q Xu, I Johansson, A Ripel, E Bodd, Jorg MorlandAbstract:: 1. The pharmacokinetics of Ethylmorphine after administration of a single dose of the cough mixture Cosylan were investigated in 10 healthy subjects. 2. The median urinary recovery of Ethylmorphine and measured metabolites was 77% over 48 h. The median tmax of unchanged Ethylmorphine was 45 min, and the terminal elimination t1/2 was 2 h. Ethylmorphine-6-glucuronide was found to be the major metabolite. 3. Two subjects had significantly lower urinary recovery (0.48 h) of morphine and morphine-glucuronides than the remainder. Furthermore, these two had urinary metabolic ratios (MRO) and partial metabolic clearances (CLmO) for O-deethylation of Ethylmorphine tentatively classifying them phenotypically as poor metabolisers of the debrisoquine/sparteine type. 4. Genotyping for cytochrome P450 (CYP) 2D6 alleles revealed five homozygote (wt/wt) and five heterozygote subjects. Two subjects phenotypically classified as poor metabolisers were genotypically CYP2D6A/wt and CYP2D6D/wt, respectively. 5. Serum and urine samples taken more than 8 and 24 h after administration of ethyl-morphine respectively, contained morphine and morphine-glucuronides, but no Ethylmorphine, Ethylmorphine-6-glucuronide or (serum only) norEthylmorphine. NorEthylmorphine could be detected after hydrolysis of urine samples in all subjects. The urinary recovery of the active metabolites morphine and morphine-6-glucuronide after administration of Ethylmorphine varied by a factor of 9 between individuals. 6. The wide variation in recovery of morphine and morphine-glucuronides after oral administration of Ethylmorphine could not be explained simply by a difference in CYP2D6 genotype. Constitutional variation in other enzymatic pathways involved in Ethylmorphine metabolism is probably crucial. Ratios of morphine to parent drug cannot be used to distinguish the source of morphine after administration of Ethylmorphine. NorEthylmorphine should be included in urine assays for opiates in forensic toxicology, and no firm conclusions about the source of morphine are possible based on serum samples obtained more than 24 h after drug administration.
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Ethylmorphine metabolism in isolated rat hepatocytes
Pharmacology & Toxicology, 1993Co-Authors: Bang Qian Xu, Tor A. Aasmundstad, Anders Bjørneboe, Asbjorg S Christophersen, Ase Ripel, Jorg MorlandAbstract:: The metabolism of Ethylmorphine has been studied in suspensions of isolated rat hepatocytes. Early during incubation, the two major metabolic intermediates detected were morphine and norEthylmorphine following N- and O-dealkylation of Ethylmorphine, respectively. During subsequent incubation the concentration of the second metabolic intermediate, normorphine increased, before the concentration peaked at approximately 20 microM (100 microM Ethylmorphine). Both morphine and normorphine were glucuronidated to form morphine-3-glucuronide and normorphine-3-glucuronide, respectively, which appeared to be the major metabolic end products. The percentage of Ethylmorphine metabolized to morphine-3-glucuronide was found to be dependent on the initial concentration of Ethylmorphine. With increasing initial Ethylmorphine concentration the relative formation of morphine-3-glucuronide was reduced (29 +/- 10% at 5 microM, 18 +/- 5% at 20 microM, and 15 +/- 4% at 100 microM mean +/- S.D., n = 10). The concentrations of Ethylmorphine and its metabolites were found to be higher in liver cells than in medium. Thus the ratios between the intra-/extra-cellular concentrations of Ethylmorphine increased somewhat from an initial value of 4 during the period for which Ethylmorphine could be detected intracellularly. The drug metabolites all exhibited ratios above 10 for the initial 100 min. of incubation. With time these ratios showed a decline, but even for prolonged incubation the ratios were 5 or higher for the end products. Thus considerable drug concentration gradients existed across the cell membrane of isolated rat hepatocytes.
F P Guengerich - One of the best experts on this subject based on the ideXlab platform.
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roles of divalent metal ions in oxidations catalyzed by recombinant cytochrome p450 3a4 and replacement of nadph cytochrome p450 reductase with other flavoproteins ferredoxin and oxygen surrogates
Biochemistry, 1995Co-Authors: Hiroshi Yamazaki, Yunefang Ueng, Tsutomu Shimada, F P GuengerichAbstract:: Recombinant cytochrome P450 (P450) 3A4 was most active in nifedipine and testosterone oxidation in a system including NADPH-P450 reductase, cytochrome b5 (b5), a semisynthetic phospholipid mixture plus cholate, glutathione, and MgCl2. The MgCl2 effect could be seen with high concentrations of Ca2+ or Sr2+ but not readily when these cations were replaced with monovalent cations. The divalent cation effect was also seen in liver microsomes. Part of the basis of this effect appears to be enhanced rates of b5 reduction, as judged from studies on deletions of reconstitution components and analysis of steady-state spectral studies. Rapid reduction of ferric P450 3A4 to ferrous was dependent upon the presence of substrate, either testosterone or Ethylmorphine. When testosterone was present, reduction was also highly dependent upon the presence of b5 and Mg2+. In the case of the substrate Ethylmorphine, the need to add b5 and Mg2+ to obtain optimal reduction rates was less pronounced. These patterns are consistent with the dramatic dependence of testosterone 6 beta-hydroxylation on b5 and the lack of dependence of Ethylmorphine N-demethylation on b5. Our interpretation is that divalent cations stimulate electron transfer from NADPH-P450 reductase to several acceptors and that substrates and b5 can bind to P450 3A4 to influence its rate of reduction by the reductase. P450 3A4 catalyzed testosterone 6 beta-hydroxylation within Escherichia coli cells. The reactions could be supported by E. coli cytosol or by purified E. coli flavodoxin and NADPH-flavodoxin reductase. Spinach ferredoxin and NADPH-ferredoxin reductase also supported catalytic activities.(ABSTRACT TRUNCATED AT 250 WORDS)