Measurement of thiopurine nucleotides in erythrocytes and clinical application to pediatric acute lymphoblastic leukemia

Abstract

Introduction: Mercaptopurine (6MP) is a purine analog. Its oral form is administered daily in maintenance therapy for pediatric acute lymphoblastic leukemia. This prodrug is absorbed and metabolized in the blood cells to 6-thioguanine (6TG) nucleotide and 6-methylmercaptopurine (6MMP) nucleotide that inhibit DNA replication and purine synthesis. In this study, those metabolites in RBC were hydrolyzed into 6TG and 6MMP using a simple preparation method, and their concentrations were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method was validated for clinical use and applied to blood samples from acute lymphoblastic leukemia (ALL) patients to investigate its effect on conventionally monitored indices to determine efficacy and compliance for therapy. Methods: Centrifuged RBC were hemolyzed and deproteinized using perchloric acid, followed by hydrolysis for 1 hour at 100℃. For liquid chromatography, C18 column was used for the stationary phase, and distilled water and acetonitrile were used for the mobile phase. In mass spectrometry, the mass transitions were 168>150.9 for 6TG and 167>125.1 for 6MMP in the positive ion mode, and isotope-substituted 6TG and 6MMP were used as internal standards. To evaluate precision of the method, two different concentrations of each drug were added to erythrocytes from three healthy persons, then measured for seven days. In order to determine the quantitative measurement limits, the experiment was repeated thrice at four low concentrations for four days. We measured the concentrations of 6TG and 6MMP in erythrocytes of children with ALL undergoing maintenance therapy for more than one month. We also analyzed whether leukocyte counts and liver enzyme levels, which were the factors determining the drug dose, could be predicted by 6MP metabolites. Results: The coefficients of variation of 6TG and 6MMP were about 5.7–8.1%, and the bias was within 5% of the target concentration. Limits of quantification with acceptable precision of 20% were set at 54 ng/mL for 6TG and 1,036 ng/mL for 6MMP. In total, 74 blood samples were collected from 37 patients for 6MP metabolite analysis after 1–26 months of maintenance therapy. Concentration of 6TG was measured in the range of 16.1 to 880 pmol/8×108 RBC and that of 6MMP was measured in the range of 55 to 20,937 pmol/8×108 RBC. There was positive correlation between 6TG and 6MMP concentrations and 6MP doses. In addition, concentration of 6TG significantly increased with patients age (r=0.448, p<0.001). Concentrations of 6MP metabolites were not correlated with neutrophil and leukocyte counts. Liver enzymes were positively correlated with both 6TG and 6MMP concentrations, showing higher ALT activity (difference 61.4 IU/L, p<0.001) in the group of 6MMP above 5,700 pmol/8×108 RBC. However, these correlations were not significant in multivariate analysis when adjusted with drug dose and duration of treatment. Conclusion: In this study, an analytical method for 6TG and 6MMP in RBC was established and applied to clinical specimens. The correlation with drug dose was good, but that with clinical indices for determining drug dose remains to be elucidated.