Pii: s0531-5131(02)00389-8

International Congress Series 1239 (2003) 589 – 592 and disposition: Toxicological significance of genotypes and phenotypes of S-mephenytoin J. Ikebuchi a,*, M. Yamada b, Y. Ogura c, Y. Yamamoto d, A. Nishimura d, K. Nishi d, K. Yamada c, Y. Irizawa a aDepartment of Legal Medicine, Faculty of Medicine, Tottori University, Nishimachi 86, Yonago 683-0826, Japan bDepartment of Human Life Science, Seibo Jogakuin Women’s College, Kyoto 612-0878, Japan cDepartment of Biochemistry, Faculty of Medicine, Tottori University, Yonago 683-0826, Japan dDepartment of Legal Medicine, Shiga University of Medical Science, Otsu 520-2192, Japan We examined the relationship between the genetic polymorphism of CYP2C19 and metabolism of omeprazole in order to assess the severity and to predict the outcome of poisoning for forensic andclinical toxicology. The genotypes observed were CYP2C19*1A (wild type), CYP2C19*2 (m1), andCYP2C19*3 (m2). The omeprazole hydroxylation index of the wild-type was À1.15, whereas for thehetero-type it was À0.78, and homo-mutated type 1.22. The genotype of CYP2C19 correlated withthe phenotype. The present results proved that genotyping assays of drug metabolizing enzymes areof great value in toxicological significance.
D 2003 Elsevier Science B.V. All rights reserved.
Keywords: CYP2C19; Genotyping; Phenotyping; Polymorphism; Toxicogenetics The 4V-hydroxylation of S-mephenytoin has been shown to be mediated by CYP2C19.
It is also important in the metabolism of a number of related hydantoins and barbiturates,as well as of structurally dissimilar drugs such as omeprazole, proguanil, and citalopram.
As a result, large interphenotypic differences occur in the disposition of these drugs, which * Corresponding author. Tel.: +81-859-34-8030; fax: +81-859-34-8076.
E-mail address: [email protected] (J. Ikebuchi).
0531-5131/03 D 2003 Elsevier Science B.V. All rights reserved.
PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 3 8 9 - 8 J. Ikebuchi et al. / International Congress Series 1239 (2003) 589–592 may affect their toxicity and efficacy. Therefore, we examined the relationship between thegenetic polymorphism of CYP2C19 and the metabolism of omeprazole in order to assessthe severity and to predict the outcome of poisoning for forensic and clinical toxicology.
In this study, we prepared DNA samples from the blood of unrelated healthy Japanese, and developed a rapid and simple genotyping method using a polymerase chain reaction(PCR)-based restriction fragment length polymorphism analysis Genotyping Fig. 1. Strategy used to genotype genomic DNA from human blood, utilizing PCR amplification of exon 5followed by SmaI digestion (CYP2C19m1) (A) and amplification of exon 4 followed by BamHI digestion(CYP2C19m2) (B). The predicted sizes of the digested DNA fragments for the various genotypes are shown.
J. Ikebuchi et al. / International Congress Series 1239 (2003) 589–592 Fig. 2. PCR analysis of exon 5 (A) and exon 4 (B) for the CYP2C19 gene. A shows the PCR amplification ofexon 5 digested with SmaI for CYP2C19m1. B shows the PCR amplification of exon 4 digested with BamHI forCYP2C19m2. The predicted sizes of the digested DNA fragments for the various genotypes are shown on theright. The sizes of the molecular weight markers (M) are shown on the left. PCR products from DNA inindividuals with the wild-type (wt) allele(s) are cleaved by the restriction enzyme (Subject Nos. 7 and 8), whereasthose in homozygous individuals with the mutation lack the SmaI or BamHI site and show a single band (SubjectNos. 1, 4 and 6). The predicted sizes of the digested DNA fragments for heterozygous individuals are shown(Subject Nos. 2, 3, 5 and 9).
procedures for the identification of CYP2C19 were performed by PCR amplification withuse of the allele-specific primers described by de Morais et al. and Kubota et al. with minor modifications. PCR products were digested with the restriction enzymes, andwere analyzed by polyacrylamide gel electrophoresis. Furthermore, CYP2C19 phenotypes Table 1Correlation between CYP2C19 genotype and phenotype a wt: Wild-type, m1: CYP2C19 mutation in exon 5, m2: CYP2C19 mutation in exon 4.
b CYP2C19 phenotype was determined by measuring omeprazole and metabolite concentrations (EM: extensive metabolizer, PM: poor metabolizer).
c Omeprazole hydroxylation index was expressed as log10 [omeprazole/5V-hydroxyomeprazole] in serum collected 2 h after omeprazole (20 mg) ingestion.
J. Ikebuchi et al. / International Congress Series 1239 (2003) 589–592 were determined by measuring omeprazole and hydroxyomeprazole concentrations in theserum, collected 2 h after omeprazole ingestion, by high-performance liquid chromatog-raphy described by Marinac et al. with minor modifications.
The genotypes observed were CYP2C19*1A (wild type: wt), CYP2C19*2 (m1), and CYP2C19*3 (m2) As shown in the omeprazole hydroxylation index ofwild-type was À1.15, whereas that of the hetero-type was À0.78, and homo-mutated type1.22. The genotype of CYP2C19 correlated with the phenotype. These results proved thatgenotyping assays of drug metabolizing enzymes could play a more important role inassessing the severity and in predicting the outcome of poisoning for forensic and clinicaltoxicology.
This work was partially supported by the Grant-in-Aid for Scientific Research, #11670416, from Japan’s Ministry of Education, Science, Sports and Culture.
[1] S.M.F. de Morais, G.R. Wilkinson, J. Blaisdell, K. Nakamura, U.A. Meyer, J.A. Goldstein, The major genetic defect responsible for the polymorphism of S-mephenytoin metabolism in humans, J. Biol. Chem. 269 (1994)15419 – 15422.
[2] S.M.F. de Morais, G.R. Wilkinson, J. Blaisdell, U.A. Meyer, K. Nakamura, J.A. Goldstein, Identification of a new genetic defect responsible for the polymorphism of (S)-mephenytoin metabolisms in Japanese, Mol.
Pharmacol. 46 (1994) 594 – 598.
[3] T. Kubota, K. Chiba, T. Ishizaki, Genotyping of S-mephenytoin 4V-hydroxylation in an extended Japanese population, Clin. Pharmacol. Ther. 60 (1996) 661 – 666.
[4] J.S. Marinac, J.D. Balian, J.W. Foxworth, S.K. Wikksie, J.C. Daus, R. Owen, D.A. Flockhart, Determination of CYP2C19 phenotype in black Americans with omeprazole, Clin. Pharmacol. Ther. 60 (1996) 138 – 144.

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