Quantification of Thiopurine Nucleotides in Erythrocytes and Clinical Application to Pediatric Acute Lymphoblastic Leukemia

Abstract

Background:Concentrations of 6-thioguanine (6TG) nucleotides and 6-methylmercaptopurine (6MMP) nucleotides in RBCs were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay was validated for clinical use and was applied to blood samples from patients taking mercaptopurine (6MP).Methods:RBCs were hemolyzed and deproteinized using perchloric acid, followed by heating for the hydrolysis of nucleotides, and the resultant base was measured using LC-MS/MS. Precision, recovery, linearity, matrix effect, and limit of quantification was validated for clinical application. Our results were compared with another institution's established LC-MS/MS assay. We measured the concentrations of 6TG and 6MMP in RBCs of pediatric patients with acute lymphoblastic leukemia (ALL), and the clinical impact of those metabolites was investigated.Results:The imprecision coefficient of variations of 6TG and 6MMP were 5.7%–8.1%, and the bias was within 5%. Lower limits of quantification were set at 54 ng/mL for 6TG and 1036 ng/mL for 6MMP. Correlation coefficients for 6TG and 6MMP were 0.997 and 1.0 in a comparison study. For clinical proof-of-concept, 74 blood samples were collected from 37 pediatric ALL patients receiving maintenance therapy. Concentration of 6TG ranged from 16.1 to 880 pmol/8 × 108 RBCs and that of 6MMP from 55 to 20,937 pmol/8 × 108 RBCs. The 6MP metabolites were not correlated with WBC or absolute neutrophil count. On the other hand, the higher 6MMP level was associated with elevated alanine aminotransferase and aspartate aminotransferase.Conclusions:In this study, an assay for the quantification of 6TG and 6MMP in RBCs was established and applied to pediatric ALL patients. Interindividual variability in 6MP metabolite concentrations was considerable and associated with elevation of liver enzymes, which may be useful in the clinical monitoring of 6MP maintenance therapy in pediatric ALL patients.

NUDT15 intermediate metabolisers are associated with lower loss of response in paediatric Crohn’s disease patients treated by combination treatment with infliximab and azathioprine

“…On the other hand, several studies have shown no significant difference in 6‐TGN levels based on NUDT15 enzyme activity 15,16 . Indeed, since erythrocytes are not the targets of thiopurines, DNA‐TG has been proposed as a more relevant thiopurine metabolite than erythrocyte‐TGN 37‐39 . Thus, the similarity in the effects of AZA when little difference exists in 6‐TGN levels between the NUDT15 variants and the normal group could not be established.…”

“…15,16 Indeed, since erythrocytes are not the targets of thiopurines, DNA-TG has been proposed as a more relevant thiopurine metabolite than erythrocyte-TGN. [37][38][39] Thus, the similarity in the effects of AZA when little difference exists in 6-TGN levels between the NUDT15 variants and the normal group could not be established. In addition, the metabolic mechanism of the NUDT15 variants might be independent of 6-TGN levels and more related to DNA-TG levels.…”

LC–MS/MS Method for Measurement of Thiopurine Nucleotides (TN) in Erythrocytes and Association of TN Concentrations With TPMT Enzyme Activity

“…Numerous methods for the analysis of thiopurine metabolites in RBCs have been published ( Erdmann et al, 1990 ; Lavi and Holcenberg, 1985 ; Lennard and Singleton, 1992 ; Moon et al, 2019 ; Thomas et al, 2018 ). Dervieux et al developed the first LC–MS/MS method for the analysis of thiopurine nucleotides in RBCs and demonstrated its clinical utility ( Dervieux et al, 2005 ).…”

Development and validation of a novel HPLC-UV method for simultaneous determination of azathioprine metabolites in human red blood cells

“…As shown in Table 1 , various analytical methods were reported for determination of 6-TGN and 6-MMPr in red blood cell [15] , [16] , [17] , [18] , [20] , [21] , [22] , [23] , [25] or whole blood [ 18 , 19 , 24 , [26] , [27] , [28] ] in patients receiving azathioprine treatment. As the most common practice at present, erythrocyte sample was precipitated by perchloric acid under the protection of dithiothreitol (DTT), with 6-TGN and 6-MMPr being acid hydrolyzed to yield 6-thioguanine (6-TG) and 6-methymercaptopurine (6-MMP) and detected by LC-UV [ [15] , [16] , [17] , [18] , [19] , [20] ] or LC-MS/MS methods [ [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] ]. However, the existing LC-UV methods often need long run time (most ≥10 min) and large volume of mobile phase solvent.…”