High-performance liquid chromatographic assays for bufuralol 1′-hydroxylase, debrisoquine 4-hydroxylase, and dextromethorphan O-demethylase in microsomes and purified cytochrome P-450 isozymes of human liver

Kronbach, Thomas; Mathys, D; Gut, Josef; Catin, Therese; Meyer, Urs

doi:10.1016/0003-2697(87)90006-6

Cited by 237 publications

(114 citation statements)

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“…7-Hydroxy-4-(trifluoromethyl)-coumarin (7-HFC, 100 M) and (3,4-difluorobenzyloxy)-5,5-dimethyl-4-(4-methylsulfonylphenyl)-(5H)-furan-2-one (DFB, 250 M), probes for UGT and CYP3A activity (Chauret et al, 1999), respectively, were incubated with the cells for 10 min. Bufuralol and tolbutamide (100 M), probes for CYP2D and 2C (Kronbach et al, 1987;Miners et al, 1988), respectively, were incubated with the cells for 2 h. Each incubation was stopped by addition of an equal volume of acetonitrile for protein precipitation. Parent compounds remaining in the incubation mixtures and metabolites were analyzed with a high-performance liquid chromatography/UV system equipped with a Waters 717 plus auto-sampler, a Waters 616 pump, a Waters 600S controller, and a Waters 996 photodiode array detector.…”

Section: Methodsmentioning

confidence: 99%

Retention of Transporter Activities in Cryopreserved, Isolated Rat Hepatocytes

Houle

Raoul²,

Lévesque³

et al. 2003

Drug Metabolism and Disposition

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:The success of cryopreservation of isolated hepatocytes with existing methodologies is assessed with respect to the retentivity of cell integrity/viability (defined by trypan blue) and metabolic activities upon thawing in comparison to those of freshly prepared cells. But the ability of the cryopreserved cells to transport xenobiotics relative to that of freshly prepared cells has not been investigated. In this study, we optimized our previous methodology for cryopreservation and evaluated the metabolism and transport of thawed hepatocytes. Half of the freshly, isolated rat hepatocytes prepared by collagenase perfusion were immediately used for studies of transport of , and tolbutamide (100 M), probes for UDP-glucuronyl transferase (UGT) and CYP3A, CYP2D, and CYP2C, respectively. The remaining half was cryopreserved using an optimized, programmed-freezing protocol, which was developed to minimize the prolonged release of latent heat during freezing. With the exception of the UGT probe, no significant difference (P > 0.05) was found in both metabolism and transport with freshly isolated versus cryopreserved hepatocytes upon thawing. In conclusion, we have demonstrated for the first time that thawed rat hepatocytes cryopreserved by a programmed-freezing protocol retain drug transport activities.

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Section: Methodsmentioning

confidence: 99%

Retention of Transporter Activities in Cryopreserved, Isolated Rat Hepatocytes

Houle

Raoul²,

Lévesque³

et al. 2003

Drug Metabolism and Disposition

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“…Published method was followed to determine dextromethorphan O-demethylation selective for CYP2D6 enzyme [18]. The incubation mixture contained NADPH-generating system…”

Section: Cyp2d6 Enzyme Assaymentioning

confidence: 99%

Is CYP2D6 phenotype predictable from CYP2D6 genotype?

Kiss

Tóth

Juhasz

et al. 2018

Microchemical Journal

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Hungary Highlights• CYP2D6 genotype-based phenotype estimation is of particular importance to personalized medication strategy.• The non-sequencing genotyping platform identified the most frequent CYP2D6 allelic variants (in 67% of the donors).• The gain-of-function mutation (-1584C>G) could explain the underestimation of CYP2D6 genotype-based prediction.• Some rare loss-of-function mutations that were not captured, could result in overestimation of CYP2D6 activity prediction.• CYP2D6 phenotype overestimation may also come from external factors modifying CYP2D6 activity or altering hepatic function.• A comprehensive genotyping and consideration of phenoconversion can improve the genotype-based CYP2D6 phenotype prediction. 2 AbstractGenetic polymorphism of cytochrome P450s results in clinically significant modifications in patients' drug metabolizing capacities. CYP2D6 has a crucial role in the elimination of several clinically important drugs (antiarrhythmics, beta-adrenergic blockers, psychopharmacons and analgesics); however, the prediction of the phenotypic appearance of CYP2D6 is a challenge. Since single nucleotide polymorphisms and gene copy number variations (gene deletion and multiplication) frequently occur in CYP2D6 gene, CYP2D6 activity particularly depends on the genetic factors.Microsomal CYP2D6 activities (dextromethorphan O-demethylation) and CYP2D6 genotypes for the most frequent allelic variants (CYP2D6*3, *4, *5, *6, *10, *41 and duplication) were determined in 128 human liver samples derived from Hungarian organ donors. Substantial inter-individual variations were observed in CYP2D6 metabolic activities that were successfully predicted from the CYP2D6 genotypes in 67% of the donors. The underestimation of CYP2D6 phenotypes in 12.5% of the donors was assumed to be originated from the overlapping ranges of CYP2D6 activity among similar diplotypes or from the presence of -1584C>G in the promoter region evoking increased transcription of the wild-type CYP2D6 allele. In an appreciable number of donors (20.3%), the genotype-based CYP2D6 phenotype prediction was overestimated because of the rare CYP2D6 allelic variants which were not included in our genotyping platform or some external factors that could alter CYP2D6 activity (medication with CYP2D6 substrate/inhibitor) and hepatic function (Augmentin therapy, chronic alcohol consumption).In conclusion, CYP2D6 genotyping for the most frequent allelic variants was able to reliably predict CYP2D6 phenotypes in most donors; however, the external factors modifying the phenotypic appearance of CYP2D6 had to be taken into account. Personalized medication strategy should include monitoring of CYP2D6 genotype in a more comprehensive manner 3 and should take external factors into consideration for an appropriate prediction of CYP2D6 metabolizing capacity.

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“…Human liver samples were obtained from organ transplantation donors and stored as described (5). Using in vitro function assays (7,13,14), 5 of the studied samples were classified as "extensive metabolizers" (debrisoquine-type phenotype) and one as "poor metabolizer". However, as these tests for cytochrome P-450 dbl activity are appropriate for extensive metabolizer phenotyping only, the phenotype of the poor metabolizer (liver #03) was confirmed by immunoquantitation of cytochrome P-450 dbl/bufl content in microsomal membranes.…”

Section: Methodsmentioning

confidence: 99%