Studies on the urinary assay of CYP-mediated endogenous corticosteroid metabolites with a LC-MS method

药物代谢酶活性的个体差异与药物反应和药物不良反应有关。分子生物学的最新进展使得从药物基因组学角度解释重要药物代谢酶，特别是细胞色素p -450 （CYPs）的部分个体间差异成为可能。然而，同样明显的是，在各自的CYP同种型中没有先前已知的功能丧失snp的受试者或患者中，仍然存在很大的个体间差异。因此，重新评估CYP异构体活性作为单个表型的兴趣激增。我们之前报道过体内CYP3A4活性可以通过尿排泄6β-羟基皮质醇（肾上腺皮质醇的CYP3A4代谢物）或其皮质醇代谢比率来评估。为了将这一想法扩展到其他被认为是由不同CYP亚型代谢的类固醇激素，我们旨在建立一种采用HPLC-UV和LC-MS的综合检测方法。我们建立了尿液中代谢分别与CYP1A2和CYP2C19相关的2-和4-羟基雌二醇和17-羟基孕酮的同时测定方法，并采用HPLC-UV法进行初步研究。我们发现，这些性类固醇代谢物的尿排泄存在很大的个体间差异，这表明有很好的机会测量这些CYP活性的个体间差异。然后，我们进一步研究建立了LC-MS方法。我们认为，这种方法将是有用的人口研究，其中大量的临床样本进行分析。

Interindividual variability of the drug metabolizing enzyme activity is associated with those of drug responses and adverse drug reactions. Recent advances in molecular biology have made it possible to account for a part of interindividual variability of important drug metabolizing enzymes, particularly cytochrome P-450s (CYPs) in the light of pharmacogenomics. Nevertheless, it has also become evident that a large interindividual variability still exists in subjects or patients having none of the previously known loss-of-function SNPs in the respective CYP isoforms. Thus, there is a surge of interest to reappraise for assessing CYP isoform activity as phenotype individually. We have previously reported that in vivo CYP3A4 activity may be assessed by urinary excretion of 6β-hydroxycortisol, a CYP3A4 metabolite of adrenal cortisol, or its metabolic ratio of cortisol. In order to extend this idea to other steroid hormones which are thought to be metabolized by different CYP isoforms, we aimed to establish a comprehensive assay method using HPLC-UV and LC-MS. We have established a simultaneous assay method for urinary 2- and 4-hydroxyestradiol and 17-hydroxyprogesterone, of which metabolism are associated with CYP1A2 and CYP2C19, respectively, with an HPLC-UV method as a preliminary study. We found that there is a large interindividual variability in the urinary excretion of those sex steroid metabolites, indicating that there is a good chance to measure interindividual variability of the these CYP activities. Then, we advanced our study to establish a LC-MS method. We consider that this method would be useful for a population study where a large number of clinical samples are to be assayed.