HPLC直接进样测定咖啡因代谢物评价三种药物代谢酶活性

目的: 建立测定尿中咖啡因的5 种主要代谢物:5-乙酰氨基-6-甲酰氨基-3-甲基尿酸(AFMU) 、1-甲基尿酸(1U) 、1-甲基黄嘌呤(1X) 、1, 7-二甲基尿酸(17U) 和1, 7-二甲基黄嘌呤(17X) 的高效液相色谱法, 以评价N -乙酰基转移酶(NAT2) 、细胞色素P450酶1A2(CYP1A2) 和黄嘌呤氧化酶(XO) 三种药物代谢酶的活性。方法: 采用反相高效液相梯度洗脱法直接进样测定尿液内咖啡因代谢产物AFMU 、1U 、1X 、17U 和17X 的相对含量, 计算AFMU /(AFMU +1X +1U) 、(AFMU +1X +1U) /17U 和1U /(1X +1U), 绘制概率分布直方图, 分别反映NAT2 、CYP1A2 和XO 的活性。结果: NAT2 活性呈两态分布, 快、慢乙酰化代谢表型的临界点为0.26, CYP1A2 和XO 酶活性呈近似正态分布。结论: 本方法简便、准确、快速,适合于尿中咖啡因代谢物的测定及NAT2 、CYP1A2和XO 等药物代谢酶活性的研究。

Determination of five metabolites of caffeine in urine to assess three drugmetabolying biotransformation enzyme activities by HPLC with direct injection method

AIM: To establish a HPLC method with direct injection for determining five major metabolites of caffeine including 5-acetylamino-6-formylamino-3-methyluracil (AFMU), 1-methylxanthine (1X), 1-methyluric acid (1U), 1, 7-dimethyluricacid (17U) and 1, 7-dimethylxanthine (17X) in the urine and assess the activities of N -acetyltransferase (NAT2), cytochrome P4501A2 (CYP1A2) and xanthine oxidase (XO). METHODS: The contents of five major metabolites of caffeine in the urine were determined by RP-HPLC method. Frequency distribution histogram were drawn by calculating the AFMU /(AFMU +1X +1U), (AFMU +1X +1U) /17U and 1U /(1X +1U) and then evaluated the activity of NAT2, CYP1A2 and XO, respectively. RESULTS: The frequency distribution histograms of NAT2 indicated two distinct distibution, and the subjects were classified as slow acetylators if PAR<0.26 or as fast if PAR >0.26.The frequency distribution of CYP1A2 and XO histograms indicated normal distribution. CONCLUSION: The method is simple, accurate, rapid, and suitable for the determination of metabolites of caffeine in urine. The method can be used to assay the activities of NAT2, CYP1A2 and XO.