Institut des Sciences Parmaceutiques et Biologiques, Pharmacie Clinique, Pharmacocinétique et Évaluation du Médicament, Université de Lyon, Université Lyon 1, Lyon, France.
Ther Drug Monit. 2012 Aug;34(4):477-80. doi: 10.1097/FTD.0b013e31825c2703.
Thiopurine drugs, widely used in cancer chemotherapy, inflammatory bowel disease, and autoimmune hepatitis, are responsible for common adverse events. Only some of these may be explained by genetic polymorphism of thiopurine S-methyltransferase. Recent articles have reported that inosine triphosphate pyrophosphatase (ITPase) deficiency was associated with adverse drug reactions toward thiopurine drug therapy. Here, we report a weak anion exchange high-performance liquid chromatography method to determine ITPase activity in red blood cells and to investigate the relationship with the occurrence of adverse events during azathioprine therapy.
ITPase activity was assessed by the enzymatic conversion of inosine triphosphate (ITP) to inosine monophosphate (IMP). The reaction was stopped by heating for 3 minutes at 120°C. IMP, inosine diphosphate, and ITP were analyzed on a Hypersil APS-2 column, a weak anion exchange phase that exhibits both ionic and hydrophobic properties.
The chromatographic method reported allows the analysis of IMP, inosine diphosphate, and ITP in a single run in <12.5 minutes. The method was linear in the range 5-1500 μmole/L of IMP. Intraassay and interassay precisions were <5% for red blood cell lysates supplemented with 50, 500, and 1000 μmole/L IMP. Km and Vmax evaluated by Lineweaver-Burk plot were 677.4 μmole/L and 19.6 μmole·L·min, respectively. The frequency distribution of ITPase from 73 patients was investigated.
The method described is useful to determine the ITPase phenotype from patients on thiopurine therapy and to investigate the potential relation between ITPase deficiency and the occurrence of adverse events.
巯嘌呤类药物广泛应用于癌症化疗、炎症性肠病和自身免疫性肝炎，会引起常见的不良反应。这些不良反应可能部分归因于巯嘌呤 S-甲基转移酶的遗传多态性。最近的文章报道称，肌苷三磷酸焦磷酸酶（ITPase）缺乏与巯嘌呤类药物治疗的药物不良反应有关。在此，我们报告了一种弱阴离子交换高效液相色谱法，用于测定红细胞中的 ITPase 活性，并研究其与巯嘌呤治疗过程中不良事件发生的关系。
通过酶促转化肌苷三磷酸（ITP）为肌苷单磷酸（IMP）来评估 ITPase 活性。反应在 120°C 加热 3 分钟终止。IMP、肌苷二磷酸和 ITP 在 Hypersil APS-2 柱上进行分析，该柱为弱阴离子交换相，具有离子和疏水性。
所报道的色谱方法允许在 <12.5 分钟内对 IMP、肌苷二磷酸和 ITP 进行单次运行分析。该方法在 5-1500 μmol/L IMP 范围内呈线性。向红细胞裂解物中添加 50、500 和 1000 μmol/L IMP 时，内和日间精密度均<5%。Lineweaver-Burk 图评估的 Km 和 Vmax 分别为 677.4 μmol/L 和 19.6 μmol·L·min。调查了 73 名患者的 ITPase 频率分布。
所描述的方法可用于确定接受巯嘌呤治疗的患者的 ITPase 表型，并研究 ITPase 缺乏与不良反应发生的潜在关系。
