Abstract：
Thiopurine is metabolized to 6-thio-(deoxy) guanosine triphosphate (6-thio-(d) GTP), which is then incorporated into DNA or RNA and causes cytotoxicity. Nudix hydrolase 15 (NUDT15) reduces the cytotoxic effects of thiopurine by converting 6-thio-(d) GTP to 6-thio-(d) guanosine monophosphate (6-thio-(d) GMP). NUDT15 polymorphisms like the Arg139Cys variant are strongly linked to thiopurine-induced severe leukocytopenia and alopecia. Therefore, measurement of NUDT15 enzymatic activity in individual patients can help predict thiopurine tolerability and adjust the dosage. We aimed to develop a quantitative assay for NUDT15 enzymatic activity in human blood samples. Blood samples were collected from donors whose NUDT15 genetic status was determined. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess the 6-thio-GTP metabolic activity in cell extracts. Because 6-thio-guanosine diphosphate (6-thio-GDP) and 6-thio-GMP were generated upon incubation of 6-thio-GTP with human blood cell extracts, the method detecting 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP was validated. All three metabolites were linearly detected, and the lower limit of quantification (LLOQ) of 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 5 μM, 1 μM, and 2 μM, respectively. Matrix effects of human blood cell extracts to detect 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 99.0 %, 100.5 %, and 101.4 %, respectively, relative to the signals in the absence of blood cell extracts. The accuracy and precision of the method and the stability of the samples were also assessed. Using this established method, the genotype-dependent differences in NUDT15 activities were successfully determined using cell extracts derived from human blood cells with NUDT15 wild-type (WT) or Arg139Cys variant and 6-thio-GTP (100 μM) as a substrate (18.1, 14.9, and 6.43 μM/h/106 cells for WT, Arg139Cys heterozygous, and homozygous variant, respectively). We developed a method for quantifying intracellular NUDT15 activity in peripheral blood mononuclear cells (PBMCs), which we defined as the conversion of 6-thio-GTP to 6-thio-GMP. Although PBMCs preparation takes some time, its reproducibility in experiments makes it a promising candidate for clinical application. This method can tell the difference between WT and Arg139Cys homozygous blood samples. Even in patients with WT NUDT15, WT samples showed variations in NUDT15 activity, which may correlate with variations in thiopurine dosage.
摘要：
硫嘌呤被代谢为6-硫代-（脱氧）鸟苷三磷酸（6-硫代-（d）GTP），然后掺入DNA或RNA并引起细胞毒性。Nudix水解酶15（NUDT15）通过将6-硫代-（d）GTP转化为6-硫代-（d）鸟苷一磷酸（6-硫代-（d）GMP）来降低硫嘌呤的细胞毒性作用。像Arg139Cys变体这样的NUDT15多态性与硫嘌呤诱导的严重白细胞减少和脱发密切相关。因此,测量个别患者的NUDT15酶活性可以帮助预测硫代嘌呤的耐受性并调整剂量。我们旨在开发一种定量测定人血液样品中NUDT15酶活性的方法。从确定了NUDT15遗传状态的供体收集血样。使用液相色谱-串联质谱（LC-MS/MS）评估细胞提取物中的6-硫代-GTP代谢活性。因为6-硫代-鸟苷二磷酸（6-硫代-GDP）和6-硫代-GMP是在将6-硫代-GTP与人血细胞提取物孵育后产生的，检测6-硫代-GTP的方法，6-硫代GDP，并验证了6-硫代-GMP。所有三种代谢物均线性检测，和6-硫代-GTP的定量下限(LLOQ)，6-硫代GDP，和6-硫代-GMP为5μM，1μM,和2μM，分别。人血细胞提取物检测6-硫代-GTP的基质效应，6-硫代GDP，6-硫代GMP为99.0%，100.5%,和101.4%，分别,相对于没有血细胞提取物的信号。还评估了方法的准确性和精密度以及样品的稳定性。使用这种既定的方法，使用来自人血细胞的细胞提取物，以NUDT15野生型（WT）或Arg139Cys变体和6-硫代GTP（100μM）作为底物（WT的18.1、14.9和6.43μM/h/106细胞，Arg139Cys杂合，和纯合变体，分别)。我们开发了一种定量外周血单核细胞（PBMC）中细胞内NUDT15活性的方法，我们将其定义为6-硫代-GTP转化为6-硫代-GMP。虽然PBMC的制备需要一些时间，其在实验中的可重复性使其成为临床应用的有希望的候选者。该方法可以区分WT和Arg139Cys纯合血液样品之间的差异。即使在患有WTNUDT15的患者中，WT样本也显示出NUDT15活性的变化，这可能与硫嘌呤剂量的变化有关。
