采用液相色谱-串联质谱法对小儿急性淋巴细胞白血病患者进行常规硫嘌呤类药物监测时对 DNA 中硫鸟嘌呤的定量分析。
Quantification of Thioguanine in DNA Using Liquid Chromatography-Tandem Mass Spectrometry for Routine Thiopurine Drug Monitoring in Patients With Pediatric Acute Lymphoblastic Leukemia.
机构信息
Department of Laboratory Medicine and Genetics, Sungkyunkwan University School of Medicine, Seoul, Korea.
Department of Laboratory Medicine, Green Cross Laboratories, Yongin, Korea.
出版信息
Ann Lab Med. 2021 Mar 1;41(2):145-154. doi: 10.3343/alm.2021.41.2.145.
BACKGROUND
We developed an assay to measure DNA-incorporated 6-thioguanine (DNA-TG) and validated its clinical applicability in Korean pediatric patients with acute lymphoblastic leukemia (ALL) in order to improve individualized thiopurine treatment and reduce the life-threatening cytotoxicity.
METHODS
The DNA-TG assay was developed based on liquid chromatography-tandem mass spectrometry, with isotope-labeled TG-d3 and guanine-d3 as internal standards. This method was applied to 257 samples of pediatric ALL patients. The DNA-TG level was compared with erythrocyte TG nucleotide (RBC-TGN) level in relation to the and genotypes, which affect thiopurine metabolism, using Spearman's rank test and repeated measure ANOVA.
RESULTS
For DNA-TG quantification, a linearity range of 10.0-5,000.0 fmol TG/μg DNA; bias for accuracy of -10.4% -3.5%; coefficient of variation for intra- and inter-day precision of 3.4% and 5.8% at 80 fmol TG/μg DNA and of 4.9% and 5.3% at 800 fmol TG/μg DNA, respectively; and recovery of 85.7%-116.2% were achieved without matrix effects or carry-over. The median DNA-TG level in the 257 samples was 106.0 fmol TG/μg DNA (interquartile range, 75.8-150.9). There was a strong correlation between DNA-TG and RBC-TGN levels (ρ=0.68, <0.0001). The DNA-TG/RBC-TGN ratio was significantly higher in NUDT15 intermediate metabolizers ( and ) than in patients with wild-type alleles (<0.0001).
CONCLUSIONS
This simple and sensitive method for measuring DNA-TG level can improve therapeutic drug monitoring for thiopurine treatment.
背景
为了改善个体化硫嘌呤治疗并降低危及生命的细胞毒性，我们开发了一种测量 DNA 整合的 6-硫代鸟嘌呤（DNA-TG）的检测方法，并在韩国儿科急性淋巴细胞白血病（ALL）患者中验证了其临床适用性。
方法
基于液相色谱-串联质谱法，我们开发了 DNA-TG 检测方法，使用同位素标记的 TG-d3 和鸟嘌呤-d3 作为内标。该方法应用于 257 例儿科 ALL 患者的 257 个样本中。通过 Spearman 秩检验和重复测量方差分析，将 DNA-TG 水平与影响硫嘌呤代谢的 和 基因型相关的红细胞 TG 核苷酸（RBC-TGN）水平进行比较。
结果
对于 DNA-TG 的定量，线性范围为 10.0-5000.0 fmol TG/μg DNA；准确度偏差为-10.4%至 3.5%；在 80 fmol TG/μg DNA 时，日内和日间精密度的变异系数分别为 3.4%和 5.8%，在 800 fmol TG/μg DNA 时，分别为 4.9%和 5.3%；回收率为 85.7%-116.2%，无基质效应或拖尾。257 个样本中 DNA-TG 的中位数为 106.0 fmol TG/μg DNA（四分位距，75.8-150.9）。DNA-TG 与 RBC-TGN 水平之间存在很强的相关性（ρ=0.68，<0.0001）。NUDT15 中间代谢物（ 和 ）的 DNA-TG/RBC-TGN 比值明显高于野生型等位基因患者（<0.0001）。
结论
该方法用于测量 DNA-TG 水平，操作简单且灵敏度高，可改善硫嘌呤治疗的治疗药物监测。
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