高效液相色谱法测定急性白血病儿童细胞色素P450 3A4活性的探讨

Detection of CYP3A4 activity with HPLC in Chinese children with acute leukemia

目的细胞色素P450 3A4(CYP3A4)参与了许多环境毒物及包括多种常用化疗药物在内的人体中50%以上的药物代谢,多态性研究表明,CYP3A4的活性具有广泛的个体差异,并显示出明显的种族和疾病分布特征.目前国内外尚无进行大样本CYP3A4活性测定方法的报道.本研究旨在建立简便、稳定、可靠的CYP3A4活性的检测方法,探讨健康儿童和急性白血病儿童CYP3A4的活性分布特点,为临床开展相关的药效学研究奠定基础.方法采用高效液相色谱法(HPLC),同时检测85名健康儿童和120名急性白血病儿童的CYP3A4活性.结果以峰面积内标法定量,线性范围0.01～5 mg/L,检测限:氢化考的松(HC)为0.01 mg/L,6β-羟基氢化考的松(6β-OHC)为0.03mg/L.HC的日内、日间误差分别小于2%和5%,6β-OHC的日内、日间误差则分别小于7%和屯10%,绝对回收率为98%～106%.健康儿童的CYP3A4活性范围为2.34～48.88,平均活性为9.76±6.99,急性髓细胞白血病(AML)患儿的CYP3A4活性为13.97±10.84,两组之间无显著性差异(P>0.05).急性淋巴细胞白血病(ALL)儿童的CYP3A4活性范围变化很大,从2.00～585.72不等,平均活性为53.52,明显高于AML组和健康儿童(P1=0.0066,P2=0.0065).结论该方法可简便、快速地测定CYP3A4活性,灵敏度和准确性较高,重复性好.ALL儿童的平均CYP3A4活性明显高于AML组和健康儿童,而后两者之间差异无显著性.

ObjectiveCytochrome P450 3A4(CYP3A4) is involved in the metabolisms of many environmental toxins as well as over 50% of drugs including commonly used chemotherapeutic drugs. Polymorphism studies have demonstrated that there are significant individual variations in CYP3A4 activity as well as various distributions in different ethnicities and diseases. Currently there was no reported method in the large scale analysis of CYP3A4 activity. This study aimed to develop a simple, stable and reliable method for the analysis of CYP3A4 activity, and to explore the distributions of the CYP3A4 activity in Chinese children. Methods High performance liquid chromatograpy (HPLC) was used to determine the CYP3A4 activity in 85 healthy children and 120 children with acute leukemia. Results The standard curve had the linear range of 0.01-5 mg/L. The minimal detectable amount was 0.01 mg/L for hydrocortisol and 0.03 mg/L for 6β-hydroxycortisol. Intra- and inter-day assay precisions for hydrocortisol were less than 2% and 5%, respectively, while those for 6β-hydroxycortisol were less than 7% and 10%,respectively. The recovery rate was 98%-106%. The activity range of CYP3A4 in the healthy controls was 2.34 - 48.88,with the average activity of 9.76±6.99. Compared with that of children with acute myelogenous leukemia (AML) (13.97±10.84), there was no statistical difference. The range of CYP3A4 activity in children with acute lymphocytic leukemia (ALL) varied from 2.00 to 585.72, with the average activity of 53.52. It was significantly higher than that of AML (P = 0.0066) and healthy children (P = 0.0065). Conclusions This method is rapid and convenient for determining CYP3A4 activity with high sensitivity,accuracy and reproducibility. ALL children have higher CYP3A4 activity than AML and healthy children, while the CYP3A4 activity was similar in the latter two groups.