Phenotyping of CYP2C19 with enantiospecific HPLC-quantification of R- and S-mephenytoin and comparison with the intron4/exon5 G-->A-splice site mutation.
S-Mephenytoin 4'-hydroxylase (CYP2C19) is a genetically polymorphic cytochrome P450. A modified method for CYP2C19 phenotyping was evaluated in 174 healthy German volunteers and the results were compared with genotyping for the intron4/exon5 G-->A splice site mutation (m1) of CYP2C19, associated with the poor metabolizer (PM) phenotype. A smaller than usual test-dose of 50 mg (R,S)-mephenytoin was used and urine samples were collected from 0 to 5 h and from 5 to 8 h after administration. Trait measurements included the mephenytoin S/R enantiomeric ratio and the hydroxylation index (i.e. the molar ratio of 4'-hydroxy-mephenytoin urinary recovery to the administered S-mephenytoin dose). S- and R-mephenytoin were quantified by isocratic HPLC with a Chiraspher column and 80% n-hexane and 20% dioxane as the mobile phase. All individuals from whom DNA was available (n = 140, including six phenotypically identified PMs) were analysed for the m1 mutation. The population frequency of this CYP2C19 mutation was 0.15. Four individuals were homozygous for m1 having S/R ratios of 0.9 or greater in both intervals of urine collection. Thus, individuals with an S/R ratio > or = 0.9 were classified as PMs and seven of all 174 phenotyped individuals were PMs (4%; 95% confidence limits: 1.6-8.1%). Heterozygous carriers of m1 (n = 34) had a median S/R ratio (5-8 h urine) of 0.06 compared to 0.01 in individuals without this mutation (n = 102; p = 0.0005, Mann-Whitney U-test). No such gene-dose relation was apparent with the hydroxylation index.(ABSTRACT TRUNCATED AT 250 WORDS)
Brockmöller
J
J
Institute of Clinical Pharmacology, Charité, Humboldt-University of Berlin, Germany.
Rost
K L
KL
Gross
D
D
Schenkel
A
A
Roots
I
I
D000225:Adenine; D000293:Adolescent; D000328:Adult; D000367:Age Factors; D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D001120:Arginine; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D001835:Body Weight; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D065731:Cytochrome P-450 CYP2C19; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers; D005091:Exons; D005260:Female; D014644:Genetic Variation; D005838:Genotype; D006147:Guanine; D006801:Humans; D007438:Introns; D008297:Male; D008617:Mephenytoin / Q000378:metabolism; D008875:Middle Aged; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010641:Phenotype; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D015203:Reproducibility of Results; D012727:Sex Characteristics; D013237:Stereoisomerism; D013379:Substrate Specificity
D017931:DNA Primers; D006147:Guanine; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19; D000225:Adenine; D003545:Cysteine; D008617:Mephenytoin