Inoue K, Yamazaki H, Imiya K, Akasaka S, Guengerich F P, Shimada T
Osaka Prefectural Institute of Public Health, Japan.
Pharmacogenetics. 1997 Apr;7(2):103-13. doi: 10.1097/00008571-199704000-00003.
Genomic DNA was isolated from livers of 39 Japanese and 45 Caucasians and the genotypes of CYP2C9 and 2C19 genes were determined with PCR methods using synthetic oligonucleotide primers. Liver microsomes were also prepared from these human samples and activities for tolbutamide methyl hydroxylation and S-mephenytoin 4'-hydroxylation were determined. The single base mutation of C416T (Arg144Cys) in CYP2C9 was detected in 22% of Caucasians but not in Japanese samples. Another single base mutation at A1061C (Ile359Leu) in the CYP2C9 gene was found with frequencies of about 8% in both races. We did not detect any individuals who have either homozygous Cys144/Cys144 or Leu359/Leu359 CYP2C9 variant nor both heterozygous Cys144-Ile359 and Arg144-Leu359 CYP2C9 variant in the human samples examined. The CYP2C19m2 genetic polymorphism was found only in Japanese people, while CYP2C19m1 type was determined in both races, with higher incidence in Japanese than in Caucasian population. Immunoblotting analysis of human liver microsomes suggested that CYP2C9 is a major component of the human CYP2C enzyme pool; it accounted for approximately 20% of total P450 in liver microsomes of both human populations. The levels of CYP2C19 protein were determined to be about 0.8% and 1.4% of total P450 (mean) in Japanese and Caucasians, respectively. We did not detect CYP2C19 protein in liver microsomes of humans who were genotyped for CYP2C19 gene as m1/m1, m1/m2, and m2/m2 variants but detected CYP2C9 protein in all of the samples examined. Good correlations were found between levels of CYP2C9 and activities of tolbutamide methyl hydroxylation (r = 0.77) and between levels of CYP2C19 and activities of S-mephenytoin 4'-hydroxylation (r = 0.86) in liver microsomes of the human samples examined. Tolbutamide methyl hydroxylation activities were lower in human samples with the Leu359 allele of CYP2C9 than those with the Cys144 allele and wild-type (Arg144-Ile359); the former type showed slightly higher K(m) values. When calculated on P450 basis, liver microsomes of individuals having m1/m1, m1/m2, and m2/m2 types of CYP2C19 had very low catalytic activities for S-mephenytoin 4'-hydroxylation. These results provide useful comparisons for pharmacokinetic and toxicokinetic models of some of the clinically used drugs that are oxidized by CYP2C proteins in humans.
从39名日本人及45名高加索人的肝脏中分离出基因组DNA，使用合成寡核苷酸引物通过PCR方法测定CYP2C9和2C19基因的基因型。还从这些人类样本中制备了肝微粒体，并测定了甲苯磺丁脲甲基羟化和S-美芬妥因4'-羟化的活性。在22%的高加索人样本中检测到CYP2C9基因中C416T（Arg144Cys）的单碱基突变，而在日本人样本中未检测到。在CYP2C9基因中发现的另一个单碱基突变A1061C（Ile359Leu）在两个种族中的频率约为8%。在所检测的人类样本中，我们未发现任何具有纯合Cys144/Cys144或Leu359/Leu359 CYP2C9变体的个体，也未发现同时具有杂合Cys144-Ile359和Arg144-Leu359 CYP2C9变体的个体。CYP2C19m2基因多态性仅在日本人中发现，而CYP2C19m1类型在两个种族中均有测定，在日本人中的发生率高于高加索人群。对人类肝微粒体的免疫印迹分析表明，CYP2C9是人类CYP2C酶库的主要成分；它在两个人群的肝微粒体中约占总P450的20%。CYP2C19蛋白水平在日本人中约占总P450（平均值）的0.8%，在高加索人中约占1.4%。在对CYP2C19基因进行m1/m1、m1/m2和m2/m2变体基因分型的人类肝微粒体中未检测到CYP2C19蛋白，但在所有检测样本中均检测到了CYP2C9蛋白。在所检测的人类样本的肝微粒体中，发现CYP2C9水平与甲苯磺丁脲甲基羟化活性之间存在良好的相关性（r = 0.77），CYP2C19水平与S-美芬妥因4'-羟化活性之间也存在良好的相关性（r = 0.86）。携带CYP2C9基因Leu359等位基因的人类样本中甲苯磺丁脲甲基羟化活性低于携带Cys144等位基因和野生型（Arg144-Ile3）的样本；前者类型显示出略高的K(m)值。以P450为基础计算时，具有m1/m1、m1/m2和m2/m2类型CYP2C19的个体的肝微粒体对S-美芬妥因4'-羟化的催化活性非常低。这些结果为人类中一些被CYP2C蛋白氧化的临床使用药物的药代动力学和毒代动力学模型提供了有用的比较。
