A convenient assay for mephenytoin 4-hydroxylase activity of human liver microsomal cytochrome P-450
Abstract
A simple and rapid method for the determination of (S)-mephenytoin 4-hydroxylase activity by human liver microsomal cytochrome P-450 has been developed. [Methyl-14C]mephenytoin was synthesized by alkylation of S-nirvanol with 14CH3I and used as a substrate. After incubation of [methyl-14C]mephenytoin with human liver microsomes or a reconstituted monooxygenase system containing partially purified human liver cytochrome P-450, the 4-hydroxylated metabolite of mephenytoin was separated by thin-layer chromatography and quantified. The formation of the metabolite depended on the incubation time, substrate concentration, and cytochrome P-450 concentration and was found to be optimal at pH 7.4. The Km and Vmax rates obtained with a human liver microsomal preparation were 0.1 mm and 0.23 nmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450, respectively. The hydroxylation activity showed absolute requirements for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH in a reconstituted monooxygenase system. Activities varied from 5.6 to 156 pmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450 in 11 human liver microsomal preparations. The basis system utilized for the analysis of mephenytoin 4-hydroxylation can also be applied to the estimation of other enzyme activities in which phenol formation occurs.
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Cited by (21)
Characterization of CYP2C19 and CYP2C9 from human liver: Respective roles in microsomal tolbutamide, S-mephenytoin, and omeprazole hydroxylations
1998, Archives of Biochemistry and BiophysicsIndividuals with drug metabolism polymorphisms involving CYP2C enzymes exhibit deficient oxidation of important therapeutic agents, includingS-mephen-ytoin, omeprazole, warfarin, tolbutamide, and nonsteroidal anti-inflammatory drugs. While recombinant CYP2C19 and CYP2C9 proteins expressed in yeast orEscherichia colihave been shown to oxidize these agents, the capacity of the corresponding native P450s isolated from human liver to do so is ill defined. To that end, we purified CYP2C19, CYP2C9, and CYP2C8 from human liver samples using conventional chromatographic techniques and examined their capacity to oxidizeS-mephenytoin, omeprazole, and tolbutamide. Upon reconstitution, CYP2C19 metabolizedS-mephenytoin and omeprazole at rates that were 11- and 8-fold higher, respectively, than those of intact liver microsomes, whereas neither CYP2C9 nor CYP2C8 displayed appreciable metabolic activity with these substrates. CYP2C19 also proved an efficient catalyst of tolbutamide metabolism, exhibiting a turnover rate similar to CYP2C9 preparations (2.0–6.4 vs 2.4–4.3 nmol hydroxytolbutamide formed/min/nmol P450). The kinetic parameters of CYP2C19-mediated tolbutamide hydroxylation (Km= 650 μM,Vmax= 3.71 min−1) somewhat resembled those of the CYP2C9-catalyzed reaction (Km= 178–407 μM,Vmax= 2.95–7.08 min−1). Polyclonal CYP2C19 antibodies markedly decreasedS-mephenytoin 4′-hydroxylation (98% inhibition) and omeprazole 5-hydroxylation (85% inhibition) by human liver microsomes. CYP2C19 antibodies also potently inhibited (>90%) microsomal tolbutamide hydroxylation, which was similar to the inhibition (>85%) observed with antibodies to CYP2C9. Moreover, excellent correlations were found between immunoreactive CYP2C19 content,S-mephenytoin 4′-hydroxylase activity (r= 0.912;P< 0.001), and omeprazole 5-hydroxylase activity (r= 0.906;P< 0.001) in liver samples from 13–17 different subjects. A significant relationship was likewise observed between microsomal tolbutamide hydroxylation and CYP2C9 content (r= 0.664;P< 0.02) but not with CYP2C19 content (r= 0.393;P= 0.184). Finally, immunoquantitation revealed that in these human liver samples, expression of CYP2C9 (88.5 ± 36 nmol/mg) was 5-fold higher than that of CYP2C19 (17.8 ± 14 nmol/mg) and nearly 8-fold higher than that of CYP2C8 (11.5 ± 12 nmol/mg). Our results, like those obtained with recombinant CYP2C enzymes, indicate that CYP2C19 is a primary determinant ofS-mephenytoin 4′-hydroxylation and low-Kmomeprazole 5-hydroxylation in human liver. Despite its tolbutamide hydroxylase activity, the low levels of hepatic CYP2C19 expression (relative to CYP2C9) may preclude an important role for this enzyme in hepatic tolbutamide metabolism and any polymorphisms thereof.
In vivo and in vitro measurement of CYP2C19 activity
1996, Methods in EnzymologyThis chapter discuses the in vivo and in vitro measurement of CYP2C 19 activity. The extensive metabolizers (EM) express CYP2C19. In poor metabolizers (PM), there is a functional deficiency in the metabolic pathway. The chapter describes the methods that primarily reflect approaches that have been found successful in laboratories. The genetic polymorphism in CYP2C19 activity has been widely studied, and the involvement of the enzyme in the metabolism of drugs other than the prototype substrate, that is, mephenytoin, continues to be of basic and applied scientific interest. The recent discovery of the molecular genetic mechanisms responsible for the PM phenotype will result in further interest, especially in the relationship between genotype and phenotype. The analytical methodologies described in the chapter have been extensively validated and widely applied, thus they provide the tools necessary for such studies.
A universal approach to the expression of human and rabbit cytochrome P450s of the 2C subfamily in Escherichia coli
1995, Archives of Biochemistry and BiophysicsHuman cytochrome P450s 2C8, 2C9, 2C18, and 2C19 and rabbit cytochrome P450s 2C1, 2C2, 2C4, 2C5, and 2C16 were expressed from their respective cDNAs inEscherichia colias chimeric enzymes in which a portion of the N-terminal membrane anchor sequence was replaced with a modified sequence derived from P450 17A. For 2C1 and 2C2 removal of the extraneous 3′-untranslated sequence allowed the successful expression of constructs that were unproductive in its presence. The levels of expression varied from 180 to 1500 nmol/liter of culture and the addition of δ-aminolevulinic acid to the culture media increased the amount of spectrally detectable P450 for several of these enzymes 2- to 10-fold. The catalytic properties of the modified human 2C P450s expressed inE. coliwere concordant with previously published data for several marker substrates including (S)-mephenytoin for P450 2C19, tolbutamide and tetrahydrocannabinol (THC) for P450 2C9, and taxol for P450 2C8. Interestingly, P450 2C19 catalyzed the 21-hydroxylation of progesterone and, to a lesser extent, catalyzed the formation of 16α-hydroxyprogesterone. The rabbit enzyme P450 2C16 catalyzed the formation of 17α- and 16α-hydroxyprogesterone in addition to 21-hydroxylation. P450 2C19 also catalyzed the methylhydroxylation of tolbu tamide and the 7-hydroxylation of THC at rates that were similar to or greater than that of P450 2C9. This work has identified important factors required for the high-level expression of 2C subfamily P450s inE. coli.The availability of these enzymes will facilitate detailed kinetic measurements for known and yet to be identified substrates.
Expression of Modified Cytochrome P450 2C10 (2C9) in Escherichia coli, Purification, and Reconstitution of Catalytic Activity
1993, Archives of Biochemistry and BiophysicsThe human cytochrome P450 (P450) 2C gene family is complex and heterologous expression methods are needed to facilitate the isolation of individual P450 proteins and the elucidation of their catalytic specificities. We prepared a series of constructs of P450 2C10 in the plasmid vector pCW, with modification of the 5′ end of the coding sequence of the cDNA. Some were not expressed at all in Escherichia coli; two were expressed at levels of 5-20 nmol membrane-bound P450 (liter culture)−1-one (2C1028) with original codons 2-7 altered by substitution of the 5′-terminal sequence described by Barnes et al. (Barnes, H. J., Arlotto, M. P., and Waterman, M. R., Proc. Natl. Acad. Sci. USA 88, 5597-5601, 1991) and one (2C1029) with original codon 2 modified, codons 3-20 deleted, and alteration of the immediate downstream codons. In both cases the P450 2C10 proteins were found essentially only in the bacterial membranes. These proteins could be purified to a high degree by solubilization and a single DEAE chromatography step. Typical P450 Fe2+ · CO absorption spectra were observed in the bacterial membranes and the purified preparations. The P450 2C1029 protein was found to have its N-terminal Met removed and the expected residues 2(Ala)-24 were identified by amino acid sequence analysis. However, the other P450 (2C1028) was apparently blocked at the N-terminus. Three native P450 2C9/10 preparations isolated from human liver showed the expected sequences (beginning with Met) for at least the first 17 residues. The blocked N-terminus in the P450 2C1028 protein may be the result of the MALLLAVF sequence, which was also used in the expression of P450 3A4 and resulted in a blocked protein. Catalytic activities of P450 2C1028 and P450 2C1029 for tolbutamide hydroxylation were similar to those measured with purified liver P450 2C9/10 in the presence of cytochrome b5, although the effect of cytochrome b5 did not always show the same pattern as with the isolated liver enzyme. The recombinant P450 2C10 enzymes did not catalyze (S)-mephenytoin 4′-hydroxylation.
TLC of Antiepileptics
2013, Thin Layer Chromatography in Drug AnalysisDrug Interaction Studies in the Drug Development Process: Studies In Vitro
2008, Handbook of Drug Metabolism, Second Edition
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On leave from Osaka Prefectural Institute of Public Health, Osaka, Japan.