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(Clinical Chemistry. 2002;48:61-68.)
© 2002 American Association for Clinical Chemistry, Inc.

HPLC Determination of Thiopurine Nucleosides and Nucleotides in Vivo in Lymphoblasts following Mercaptopurine Therapy

Thierry Dervieux1, Yaqin Chu1, Yi Su1, Ching-Hon Pui2,3, William E. Evans1,3 and Mary V. Relling1,3a

Departments of
1 Pharmaceutical Sciences and
2 Hematology-Oncology, St. Jude Children’s Research Hospital, Memphis, TN 38105
3 College of Pharmacy and Medicine, University of Tennessee, Memphis, TN 38163

aAddress correspondence to this author at: Department of Pharmaceutical Sciences, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105. Fax 901-525-6869; e-mail mary.relling{at}stjude.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Mercaptopurine is a prodrug requiring intracellular activation to thiopurine nucleotides to exert antileukemic effect. We developed a reversed-phase liquid chromatographic assay for the quantification of mercaptopurine, thioguanine, and methylmercaptopurine nucleoside and nucleotide concentrations in the target tissue, the leukemic lymphoblast.

Methods: Leukemic blasts were isolated from peripheral blood and bone marrow by a standard Ficoll-hypaque procedure. Proteins were removed by ultrafiltration in the presence of dithiothreitol. Thiopurine ribonucleotides were converted into their respective ribonucleosides by treatment of ultrafiltrate with acid phosphatase. Thiopurine nucleosides and bases were measured by direct injection of ultrafiltrate into the chromatographic system. Thiopurine nucleotide concentrations were calculated by subtracting the thiopurine nucleoside concentrations measured after treatment with acid phosphatase from those measured after direct injection of ultrafiltrate in the chromatographic system. Analytes were separated on a C18 Supelco column with ammonium phosphate-methanol eluent coupled with ultraviolet detection.

Results: CVs for intra- and interday precision were 1.1–14% (median, 4.9%), and recovery of added analyte was 89–126% (median, 105%) at low and high concentrations of analytes, except for mercaptopurine riboside. The median signal for each of the five metabolites in lymphoblast samples was 98% (range, 80–106%) of that in water. Detection limits for thiopurine bases and nucleosides ranged from 0.5 to 4.5 pmol/5 x 106 cells.

Conclusions: This method is suitable for measurement of thiopurine metabolite concentrations in lymphoblasts in children with acute lymphoblastic leukemia following a single dose of intravenous mercaptopurine.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mercaptopurine (MP), 1 the analog of hypoxanthine, is an antileukemic agent widely used to treat acute lymphoblastic leukemia (ALL) (1). MP is a prodrug requiring intracellular activation to thiopurine nucleotides to exert cytotoxicity. Intracellularly, MP is converted by hypoxanthine guanine phosphorybosyltransferase into thioinosine monophosphate (TIMP) and subsequently into thioguanosine monophosphate by a two-step process involving inosine monophosphate dehydrogenase and guanosine monophosphate synthetase (2). This process is in competition with methylation by thiopurine methyltransferase, an enzyme subject to genetic polymorphism (3)(4). Thiopurine methyltransferase converts MP into inactive methylmercaptopurine (MMP), but also metabolizes TIMP into active methyl-TIMP (5)(6)(7). Each of the thiopurine riboside monophosphates (TIMP, thioguanosine monophosphate, and methyl-TIMP) can be converted into thiopurine ribonucleotide triphosphates (8)(9) and dephosphorylated into thiopurine ribonucleosides [mercaptopurine riboside (rMP), mercaptoguanosine riboside (rTG), and MMP riboside (rMMP)] by 5'-nucleotidase (10)(11).

In clinical practice, studies have reported that the antileukemic effect of MP is related to the formation of thioguanine nucleotides (TGNs) (12)(13)(14), which are incorporated into genomic DNA in their reduced form (deoxythioguanosine) (15). However, MMP nucleotides (MeMPNs) are potent inhibitors of de novo purine synthesis in vitro and, therefore, can contribute to the effects of MP (16)(17). The monitoring of MP therapy with quantification of thiopurine metabolite concentrations by HPLC in plasma (18)(19) or erythrocytes (14)(20)(21) has been advocated by several groups as a strategy to optimize therapy. Although erythrocytes are a convenient tissue to study the intracellular metabolism of MP, very little is known about the disposition of thiopurine metabolites in the target of MP therapy, the leukemic blast. Several chromatographic methods for the determination of intracellular thiopurine nucleotide concentrations in erythrocytes (22)(23)(24) or peripheral lymphoblasts (8)(9)(26)(27) have been developed. Many of these methods are based on acid hydrolysis of the thiopurine nucleotide moieties to thiopurine bases, making them incapable of distinguishing among nucleobases, nucleosides, and nucleotides (23)(24)(25)(26). In contrast, other methods have proposed direct quantification of thiopurine mono-, di-, or triphosphate nucleotides by ion-pairing chromatography (8)(9)(22), but they require calibrators that are not available commercially. Here we report a HPLC method sensitive enough to measure thiopurine nucleosides and nucleotides in the target tissue, the leukemic lymphoblast, after MP therapy given to children with ALL.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
chemicals and preparation of calibrators
MP, rMP, rTG, MMP, rMMP, D,L-dithiothreitol (DTT), and acid phosphatase (type VII from white potato) were obtained from Sigma. TIMP and thioguanosine diphosphate (TGDP) were kindly provided by the National Cancer Institute (Bethesda, MD). Sodium citrate and monobasic ammonium phosphate were purchased from Fisher Scientific. HPLC-grade methanol was purchased from Baxter Healthcare. Microcon 10 ultrafiltration concentrators were from Amicon.

MP was dissolved in 0.1 mol/L sodium hydroxide, rTG was dissolved in 0.001 mol/L sodium hydroxide, rMMP was dissolved in 0.1 mol/L phosphate buffer (pH 7), and rMP and MMP were dissolved in 0.1 mol/L hydrochloric acid. Thiopurine nucleotides (TIMP and TGDP) were dissolved in water containing 10 mmol/L DTT. After the compounds were dissolved, the final concentrations were confirmed with a Shimadzu UV-1601 spectrophotometer; the molar absorptivities at the given wavelengths were as follows: MP, {epsilon}312 nm = 19 600 at pH 12; rMP, {epsilon}322 nm = 22 500 at pH 1.0; rTG, {epsilon}319 nm = 21 000 at pH 5.0; MMP, {epsilon}295 nm = 16 200 at pH 1.0; rMMP, {epsilon}289 nm = 19 050 at pH 7.0); TIMP, {epsilon}322 nm = 27 600 at pH 4.6; TGDP, {epsilon}342 nm = 26 700 at pH 4.6 [communication from Sigma and Ref. (28)]. The above aqueous solutions were diluted at a final concentration of 100 mg/L in 0.1 mol/L hydrochloric acid (thiopurine nucleosides and bases) or water (TIMP and TGDP) containing 10 mmol/L DTT and stored at -70 °C. Calibrators were stable for at least 6 months under these conditions.

To check the efficacy of acid phosphatase hydrolysis of MeMPNs, the nucleotides were synthesized by incubating CEM cells (Human T lineage leukemia cells) with MMP riboside at a final concentration of 10 µmol/L for 24 h. After incubation, cells were centrifuged, washed with phosphate-buffered saline, and resuspended in water at a final concentration of 100 x 106 cells/mL. Subsequently, the cell suspension was sonicated, transferred into ultrafiltration concentrators, and centrifuged for 1 h at 12 000g. The ultrafiltrate was stored at -20 °C. Concentrations of MeMPNs present in the ultrafiltrate were determined by an acid hydrolysis procedure as described (25). Briefly, 300 µL of cell extract was heated for 1 h at 100 °C in the presence of 30 µL of 700 mL/L perchloric acid to allow the conversion of MeMPNs to 4-amino 5-methylthiocarbonyl imidazole (29). Concentrations were determined by comparison with a calibration curve for rMMP treated using the same conditions. The final concentration of MeMPNs in the cell extract was 54 µmol/L. MMP and rMMP were undetectable in the prepared ultrafiltrate.

hplc instrument and chromatographic separation of compounds
The liquid chromatography system consisted of an automated system with two Shimadzu LC-10AS liquid chromatography pumps, a SCL-10A system controller, a SIL-10A autoinjector, a sample cooler kept at 4 °C, and a SPS-10A ultraviolet–visible (UV-Vis) detector. Chromatograms were acquired and analyzed with the Shimadzu Class VP 5.0 software running on a Dell computer.

The HPLC separation (30) was performed on a 25 cm x 4.6 mm Supelco column (5-µm particle size), protected by a Supelco guard column. Mobile phase A consisted of 98% monobasic ammonium phosphate (30 mmol/L, pH 2.90) and 2% methanol containing 0.26 mmol/L DTT. Mobile phase B consisted of 60% monobasic ammonium phosphate (30 mmol/L, pH 2.90) and 40% methanol. The samples were isocratically eluted at a flow rate of 1 mL/min, with 12% mobile phase B for 15 min and a 25-min linear gradient to 100% phase B. After 40 min, the mobile phase was returned to 12% mobile phase B. One sample was injected every 60 min. Detection was at 330 nm for the first 30 min (detection of MP, rMP, and rTG) and at 290 nm (MMP and rMMP) thereafter.

calibrators and calibration curves
Calibration curves were prepared by adding known amounts of MP, rMP, rTG, MMP, and rMMP to a pool of lymphoblasts isolated from patients with ALL before any chemotherapy. We anticipated low metabolite concentrations. The range for these enriched calibrators were as follows: MP, 0.8–13.1 pmol/5 x 106 cells; rMP, 0.9–14.1 pmol/5 x 106 cells; rTG, 0.8–13.4 pmol/5 x 106 cells; MMP, 7.5–120.3 pmol/5 x 106 cells; and rMMP, 4.2–67.0 pmol/5 x 106 cells. All enriched calibrators were assayed in duplicate. Calibration curves were fit by linear regression using peak heights vs concentrations.

precision, recovery, and extraction efficiency
Intra- and interday precision and recoveries were determined by analyzing enriched low and high concentrations of thiopurines added to pretreatment lymphoblasts prepared independently from the calibrators. Intraday analysis was performed with 10 enriched replicates, whereas interday evaluation was assessed with 3 replicates on 5 different days. Recovery was calculated as the measured concentrations in the enriched samples relative to the target concentration (measured concentration/target concentration x 100%). Imprecision was determined by the CV. Precision and recovery were also determined from three replicates of unknown samples enriched with low, medium, and high concentrations of thiopurines with the analyst blinded to the sample preparation and concentrations.

To estimate extraction efficiencies of MP, rMP, rTG, MMP, and rMMP, we compared the peak heights from enriched lymphoblast samples with those from samples prepared with water at the same concentrations within the validated range.

The conversion of thiopurine nucleotides to thiopurine nucleosides was determined with available thiopurine nucleotide calibrators (TIMP and TGDP) and with MeMPNs that were formed after incubating CEM cells with rMMP, as described above. TIMP, TGDP, and MeMPNs were added to lymphoblasts at concentrations of 100–500 pmol/5 x 106 cells. Precision and recovery were determined after quantification of rMP, rTG, or rMMP concentrations formed after enzymatic treatment (with five replicates at each concentration 4 different days). The activity of each lot of acid phosphatase was tested using a pool of patient lymphoblasts following treatment with MP.

sample treatment procedure
Cell pellets (5 x 106) resuspended in a final volume of 200 µL of 10 mmol/L sodium citrate (pH 4.5) were sonicated for 10 s, and 10 µL of a 10 mmol/L DTT solution was added. The sample mixture was transferred to a Microcon-10 and centrifuged 30 min at 12 000g. Ultrafiltrates were separated into two aliquots of 90 µL each. To quantify thiopurine nucleotides, a total of 2.5 µL of acid phosphatase was added to the first aliquot, and the mixture was heated for 2 h at 56 °C to convert thiopurine ribonucleotides into thiopurine ribonucleosides. Thioinosine nucleotides were converted into rMP; TGNs into rTG, and MeMPNs into rMMP. After incubation and cooling, 5.0 µL of 10 mmol/L DTT was added to the mixture and centrifuged for 2 min. A total volume of 80 µL was injected into the HPLC system. To quantify thiopurine nucleosides and bases (MP, rMP, rTG, MMP, and rMMP), the second aliquot of ultrafiltrate was not treated with acid phosphatase. A total of 2.5 µL of water and 5.0 µL of 10 mmol/L DTT were added to the ultrafiltrate, which was then centrifuged for 2 min; a total volume of 80 µL was injected into the HPLC system.

Thiopurine nucleotide concentrations (MPNs, TGNs, and MeMPNs) were calculated by subtracting the thiopurine nucleoside concentrations measured after direct injection of ultrafiltrate into the chromatographic system from those measured after treatment with acid phosphatase.

application to patient samples
Blood samples (5 mL) were collected into heparin-containing tubes before and 6 and 20 h after the initiation of a 6-h intravenous infusion (1 g/m2) of MP in children with newly diagnosed ALL after written informed consent was obtained from the guardians or parents. Bone marrow aspirates were drawn 20 h after initiation of MP infusion. Lymphoblasts were isolated using a Ficoll-hypaque density gradient procedure and washed three times with a solution of HEPES, Hank’s buffered salt solution, and heparin. The final cell yield was determined by hemocytometer and the percentage of viability by trypan blue exclusion. The mononuclear cells layer contained, on average, 10% red blood cells. Peripheral blood red cell lysates were prepared as described previously (31). Thiopurine metabolites were measured as described for blasts in 100 samples. The correlation coefficient between total concentrations measured in the Ficoll-hypaque mononuclear layer and those calculated after subtracting the concentrations arising from red blood cells was 0.967 (slope, 1.079) for TGNs and 0.929 (slope, 1.14) for MeMPNs, demonstrating that the residual red blood cells had little effect on the estimate of blast concentrations. Mononuclear cells were pelleted (5.0 x 106 to 20 x 107 cells depending on cell yield) and stored at -80 °C until analysis. Results were normalized to 5 x 106 mononuclear cells. Patient results are expressed as mean ± SE. Intrapatient comparisons of thiopurine metabolite concentrations were assessed using the Wilcoxon test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
separation of compounds, calibration curves, and detection limit
Typical chromatograms of 5 x 106 pretreatment bone marrow lymphoblasts to which thiopurines had been added are presented in Fig. 1 . Calibration curves demonstrated a linear relationship between peak height and concentration for all analytes, with correlation coefficients >0.995 for all. Equations describing the calibration curves were as follows: MP, y = 0.0169x - 0.2442; rMP, y = 0.0226x - 0.6170; rTG, y = 0.0232x - 0.2919; MMP, y = 0.0207x + 0.0720; rMMP, y = 0.0173x - 0.0849, where y is the peak height and x is the concentration of added thiopurine.



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  		Figure 1. Chromatogram of prechemotherapy lymphoblasts before (A) and after (B) addition of analytes.

Retention times were as follows: MP, 8.0 min; rMP, 12.1 min; rTG, 12.9 min; MMP, 33.4 min; rMMP, 37.0 min. TIMP and TGDP calibrators are eluted at 4.9 and 5.0 min, respectively (not shown). Concentrations of the analytes were as follows: MP (9.9 pmol/5 x 106 cells), rMP (10.5 pmol/5 x 106 cells), rTG (10.0 pmol/5 x 106 cells), MMP (90.2 pmol/5 x 106 cells), and rMMP (50.3 pmol/5 x 106 cells).

The limits of detection, defined as three times the signal-to-noise ratio, were 0.5 pmol/5 x 106 cells for MP, rMP, and rTG and 4.5 and 2.5 pmol/5 x 106 cells for MMP and rMMP, respectively. The precolumn was changed every 30 injections, and the analytical column demon-strated no deterioration in performance after up to 300 injections.

precision, recovery, and extraction efficiencies
The intra- and interday precision and recoveries for the assay are summarized in Table 1 . The CVs for intra- and interday precision were <15% at low and high concentrations of analytes. Recovery of analytes added to samples was 89–126% for MP, rTG, MMP, and rMMP. However, at low concentrations of rMP (1.1 pmol/5 x 106 cells), interday recoveries were poor because of an interfering peak in some pretherapy samples. Mean extraction efficiencies were 97.0%, 99.7%, 96.5%, 80.3%, and 85.3% at low concentrations and 103.3%, 105.8%, 98.8%, 105.9%, and 105.5% at high concentrations of MP, rMP, rTG, MMP, and rMMP, respectively. For three replicates of "unknown" lymphoblast samples enriched with different concentrations of compounds, with the analyst blind to the sample concentrations, median imprecision (as CVs) and recovery were 4.2% (range, 0.4–14%) and 102% (range, 87–114%), respectively.


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  		Table 1. Precision and recoveries for thiopurine metabolites in leukemic blasts.

The efficiency of dephosphorylation of TIMP, TGDP, and MeMPNs after treatment with acid phosphatase was determined at concentrations of 100–500 pmol/5 x 106 cells. After dephosphorylation, TIMP and TGDP peaks were undetectable in the chromatograms, whereas rMP and rTG appeared (not shown). Similarly, no rMMP peak was detectable before dephosphorylation of MeMPN calibrators added to lymphoblasts, whereas the rMMP peak appeared after conversion with acid phosphatase. Complete conversion of TIMP, TGDP, and MeMPNs to rMP, rTG, and rMMP, respectively, was demonstrated by the recoveries >90% and CVs <15% for TIMP, TGDP, and MeMPNs (Table 2 ).


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  		Table 2. Efficacy of acid phosphatase conversion of nucleotides to nucleosides.1

The activity of the acid phosphatase was tested, for each lot purchased, against a pool of lymphoblasts from patients treated with MP. Over a period of 1 year, the variability of rTG and rMMP measured after enzymatic conversion in this control pool was 13.4% and 6.2%, respectively (n = 7).

application to patient samples
The method was applied to the in vivo assessment of lymphoblast thiopurine metabolite concentrations 6 and 20 h after initiation of a 1 g/m2 intravenous MP infusion given over 6 h to children with newly diagnosed ALL. Typical chromatograms of bone marrow lymphoblasts of a patient at 20 h after initiation of MP infusion are presented in Fig. 2 .



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  		Figure 2. Chromatogram of bone marrow lymphoblasts 20 h after initiation of a 1 g/m2 MP infusion.

(A), thiopurine metabolite concentrations in ultrafiltrate without acid phosphatase treatment. Concentrations were as follows: MP, 2.6 pmol/5 x 106 cells; rMP, 3.2 pmol/5 x 106 cells; rTG, 4.0 pmol/5 x 106 cells; MMP, none detected; rMMP, 10.3 pmol/5 x 106 cells. Note the presence of thioinosine and thioguanine nucleotides coeluting in the chromatographic system at 4.9 min (arrow) before treatment with acid phosphatase. (B), thiopurine metabolite concentrations measured after treatment of ultrafiltrate with acid phosphatase. Concentrations were as follows: MP, 2.8 pmol/5 x 106 cells; rMP, 10.6 pmol/5 x 106 cells; rTG, 20.0 pmol/5 x 106 cells; MMP, none detected; rMMP, 35.8 pmol/5 x 106 cells. Calculated thiopurine nucleotide concentrations were as follows: MPNs, 7.4 pmol/5 x 106 cells; TGNs, 16.0 pmol/5 x 106 cells; MeMPNs, 25.5 pmol/5 x 106 cells. Note the absence of thioinosine and thioguanine nucleotides eluting at 4.9 min (arrow) and the increase in peak heights of the respective nucleosides after acid phosphatase treatment compared with no treatment with acid phosphatase (chromatogram A).

The concentrations of TGNs, MeMPNs, rTG, and rMMP measured in bone marrow mononuclear cells are shown in Fig. 3A . At 20 h, TGNs were undetectable in only 2 of 41 samples. Thiopurine base, nucleoside, and nucleotide concentrations measured at 6 and 20 h in bone marrow mononuclear cells from 20 patients with peripheral lymphoblasts are presented in panels B, C, and D of Fig. 3 , respectively. At 6 h, MPN metabolites predominated over TGN and MeMPN metabolites, whereas at 20 h, a decrease in MPN concentrations (P <0.001) was associated with an increase in MeMPN concentrations (P <0.001) without significant change of TGN concentrations (P = 0.16). The total thiopurine nucleotide concentrations (MPN + TGN + MeMPN) were similar at 6 and 20 h (P = 0.48).



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  		Figure 3. Thiopurine metabolite concentrations measured at 6 and 20 h after 1 g/m2 MP infusion in children with ALL.

Results are expressed as mean ± SE (error bars). (A), mean TG and MMP nucleoside and nucleotide concentrations measured at 20 h in bone marrow lymphoblasts in 41 children. (B), mean thiopurine nucleoside concentrations measured at 6 and 20 h in peripheral lymphoblasts in 20 children. Thiopurine nucleosides were measured after direct injection of ultrafiltrate into the chromatographic system. (C), mean thiopurine nucleotide concentrations measured at 6 and 20 h in peripheral lymphoblasts in 20 children. Thiopurine nucleotide concentrations were calculated by subtraction of thiopurine nucleoside concentrations measured after direct injection of ultrafiltrate from the total thiopurine nucleoside concentrations after treatment with acid phosphatase. (D), mean total thiopurine nucleoside and nucleotide concentrations measured at 6 and 20 h in peripheral lymphoblasts in 20 children. IV, intravenous.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although MP has been administered for more than 40 years in the treatment of ALL, very little is known about the in vivo intracellular disposition of thiopurine metabolites in the target of MP therapy, the leukemic blast. Two different strategies have been reported for the quantification of thiopurine nucleotide concentrations in lymphoblasts or peripheral mononuclear cells (8)(9)(26)(27). These strategies use protein precipitation with acid in the presence of DTT, followed by acid hydrolysis and heating to convert thiopurine nucleotides into thiopurine bases (26), or protein-free samples are directly injected and thiopurine nucleotides are detected by ion-pairing chromatography (8)(9)(27). Each of these approaches has limitations. The acid hydrolysis procedure cannot establish which portion of the thiopurine base originates from the thiopurine nucleotide or thiopurine nucleoside, or is present as the base. In contrast, direct detection of thiopurine nucleotides by ion-pairing chromatography has the advantage that it quantifies thiopurine nucleotide mono-, di-, or triphosphates; however, these calibrators are not available commercially. In addition, measuring each of the thiopurine mono-, di-, or triphosphates compromises the sensitivity to detect nucleotides vs other thiopurine metabolites.

Our method strikes a compromise in which we differentiate among thiopurine bases, nucleosides, and nucleotides, but do not separate nucleotides into mono-, di-, or triphosphates. We removed proteins by ultrafiltration and then enzymatically converted thiopurine nucleotides into thiopurine nucleosides with acid phosphatase. Cleavage of the ribonucleoside-phosphate bonds during the enzymatic treatment liberates the thiopurine nucleoside moiety, which is detected in our chromatographic system by an ultraviolet detector. Because the ultrafiltration step removes all proteins, it prevents the possible ex vivo catabolism of the thiopurine nucleotides by other purine enzymes, such as purine nucleotide phosphorylase. This method is rapid, robust, and accurate for the concentrations present in vivo in leukemic blasts, using only 5 x 106 cells. The method is linear for all analytes, with intra- and interday CVs <15% for both low and high concentrations. However, an endogenous peak that coelutes with rMP did not allow an exact measurement of low concentrations of this metabolite.

The low number of cells required in this assay makes our method suitable for use in pediatric patients, in whom blood volume and bone marrow samples are usually limited. We measured bone marrow and peripheral blast thiopurine metabolites after administration of intravenous MP. To our knowledge, such a study has never been conducted in a large number of patients and never in bone marrow blasts. We observed substantial interpatient differences in thiopurine metabolite concentrations, which could have been attributable to interindividual differences in enzymes such as thiopurine methyltransferase or 5'-nucleotidase (10)(11)(31).

MPNs predominated over thioguanine and MeMPN concentrations at 6 h, but at 20 h, MPN concentrations were lower and MeMPN concentrations were higher relative to concentrations at 6 h. Total thiopurine nucleotide concentrations measured at 6 and 20 h were similar. This suggests that MPNs were preferentially methylated by thiopurine methyltransferase between 6 and 20 h, rather than converted by inosine monophosphate dehydrogenase and guanosine monophosphate synthetase into TGNs. Previous in vitro reports have shown that TIMP can inhibit inosine monophosphate dehydrogenase (32)(33). Our in vivo results are consistent with these observations: inhibition of inosine monophosphate dehydrogenase by TIMP or related nucleotides at 6 h may have limited the subsequent formation of TGNs, leaving TIMP available for methylation to methylthioinosine monophosphate.

In conclusion, we have developed a sensitive and specific reversed-phase chromatographic method for the determination of lymphoblast thiopurine bases, nucleosides, and nucleotides. The method is accurate and suitable for clinical pharmacologic studies of MP in children with ALL.


   Acknowledgments
 
This work was supported by Grants CA 36401, CA 78224, and CA21765 from the NIH; by a Center of Excellence grant from the State of Tennessee; and by American Lebanese Syrian Associated Charities (ALSAC). We thank our clinical staff for scrupulous attention to patient care and management of blood sampling; our research nurses, Sheri Ring, Lisa Walters, Terri Kuehner, Margaret Edwards, and Paula Condy; and the patients and their parents for their participation in this study.


   Footnotes
 
1 Nonstandard abbreviations: MP, 6-mercaptopurine; ALL, acute lymphoblastic leukemia; TIMP, thioinosine monophosphate; MMP, methylmercaptopurine; rMP, MP riboside; rTG, thioguanine riboside; rMMP, MMP riboside; TGN, thioguanine nucleotide; DTT, dithiothreitol; TGDP, thioguanosine diphosphate; MeMPN, MMP nucleotide; and MPN, mercaptopurine nucleotide.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pui CH, Evans WE. Acute lymphoblastic leukemia. N Engl J Med 1998;339:605-615.[Free Full Text]
  2. Elion GB. The purine path to chemotherapy. Science 1989;24:441-447.
  3. Weinshilboum RM, Sladek SL. Mercaptopurine pharmacogenetics: monogenic inheritance of erythrocyte thiopurine methyltransferase activity. Am J Hum Genet 1980;32:651-662.[ISI][Medline] [Order article via Infotrieve]
  4. Relling MV, Hancock ML, Rivera GK, Sandlund JT, Ribeiro RC, Krynetski EY, et al. Mercaptopurine therapy intolerance and heterozygosity at the thiopurine S-methyltransferase gene locus. J Natl Cancer Inst 1999;91:2001-2008.[Abstract/Free Full Text]
  5. Deininger M, Szumlanski CL, Otterness DM, Van Loon J, Ferber W, Weinshilboum RM. Purine substrates for human thiopurine methyltransferase. Biochem Pharmacol 1994;48:2135-2138.[ISI][Medline] [Order article via Infotrieve]
  6. Krynetski EY, Krynetskaia NF, Yanishevski Y, Evans WE. Methylation of mercaptopurine, thioguanine, and their nucleotide metabolites by heterologously expressed human thiopurine S-methyltransferase. Mol Pharmacol 1995;47:1141-1147.[Abstract]
  7. Bokkerink JP, Stet EH, De Abreu RA, Damen FJ, Hulscher TW, Bakker MA, van Baal JA. 6-Mercaptopurine: cytotoxicity and biochemical pharmacology in human malignant T-lymphoblasts. Biochem Pharmacol 1993;45:1455-1463.[ISI][Medline] [Order article via Infotrieve]
  8. Zimmerman TP, Chu LC, Bugge CJ, Nelson DJ, Lyon GM, Elion GB. Identification of 6-methylmercaptopurine ribonucleoside 5'-diphosphate and 5'-triphosphate as metabolites of 6-mercaptopurine in man. Cancer Res 1974;34:221-224.[Abstract/Free Full Text]
  9. Keuzenkamp-Jansen CW, De Abreu RA, Bokkerink JP, Trijbels JM. Determination of extracellular and intracellular thiopurines and methylthiopurines by high-performance liquid chromatography. J Chromatogr B Biomed Appl 1995;672:53-61.[ISI][Medline] [Order article via Infotrieve]
  10. Pieters R, Huismans DR, Veerman AJ. Are children with lymphoblastic leukaemia resistant to 6-mercaptopurine because of 5'-nucleotidase?. Lancet 1987;2:1471.[ISI][Medline] [Order article via Infotrieve]
  11. Kerstens PJ, Stolk JN, Hilbrands LB, van de Putte LB, De Abreu RA, Boerbooms AM. 5-Nucleotidase and azathioprine-related bone-marrow toxicity. Lancet 1993;342:1245-1246.[ISI][Medline] [Order article via Infotrieve]
  12. Lennard L, Lilleyman JS. Variable mercaptopurine metabolism and treatment outcome in childhood lymphoblastic leukemia. J Clin Oncol 1989;7:1816-1823.[Abstract]
  13. Evans WE, Horner M, Chu YQ, Kalwinsky D, Roberts WM. Altered mercaptopurine metabolism, toxic effects, and dosage requirement in a thiopurine methyltransferase-deficient child with acute lymphocytic leukemia. J Pediatr 1991;119:985-989.[ISI][Medline] [Order article via Infotrieve]
  14. Lennard L, Lilleyman JS, Van Loon J, Weinshilboum RM. Genetic variation in response to 6-mercaptopurine for childhood acute lymphoblastic leukaemia. Lancet 1990;336:225-229.[ISI][Medline] [Order article via Infotrieve]
  15. Warren DJ, Andersen A, Slordal L. Quantitation of 6-thioguanine residues in peripheral blood leukocyte DNA obtained from patients receiving 6-mercaptopurine-based maintenance therapy. Cancer Res 1995;55:1670-1674.[Abstract/Free Full Text]
  16. Vogt MH, Stet EH, De Abreu RA, Bokkerink JP, Lambooy LH, Trijbels FJ. The importance of methylthio-IMP for methylmercaptopurine ribonucleoside (Me-MPR) cytotoxicity in Molt F4 human malignant T-lymphoblasts. Biochim Biophys Acta 1993;1181:189-194.[Medline] [Order article via Infotrieve]
  17. Dervieux T, Blanco JG, Krynetski EY, Vanin E, Roussel MF, Relling MV. Differing contribution of thiopurine methyltransferase to mercaptopurine versus thioguanine effects in human leukemic cells. Cancer Res 2001;61:5810-5816.[Abstract/Free Full Text]
  18. Koren G, Ferrazini G, Sulh H, Langevin AM, Kapelushnik J, Klein J, et al. Systemic exposure to mercaptopurine as a prognostic factor in acute lymphocytic leukemia in children. N Engl J Med 1990;323:17-21.[Abstract]
  19. Sulh H, Koren G, Whalen C, Soldin S, Zipursky A, Greenberg M. Pharmacokinetic determinants of 6-mercaptopurine myelotoxicity and therapeutic failure in children with acute lymphoblastic leukemia. Clin Pharmacol Ther 1986;40:604-609.[ISI][Medline] [Order article via Infotrieve]
  20. Relling MV, Hancock ML, Boyett JM, Pui C-H, Evans WE. Prognostic importance of 6-mercaptopurine dose intensity in acute lymphoblastic leukemia. Blood 1999;93:2817-2823.[Abstract/Free Full Text]
  21. Dervieux T, Medard Y, Verpillat P, Guigonis V, Duval M, Lescoeur B, et al. Possible implication of thiopurine S-methyltransferase in the occurrence of infectious episodes during maintenance therapy of acute leukemia with mercaptopurine. Leukemia 2001;15:1706-1712.[ISI][Medline] [Order article via Infotrieve]
  22. Lavi LE, Holcenberg JS. A rapid and sensitive high-performance liquid chromatographic assay for 6-mercaptopurine metabolites in red blood cells. Anal Biochem 1985;144:514-521.[ISI][Medline] [Order article via Infotrieve]
  23. Erdmann GR, France LA, Bostrom BC, Canafax DM. A reversed phase high performance liquid chromatography approach in determining total red blood cell concentrations of 6-thioguanine, 6- mercaptopurine, methylthioguanine, and methylmercaptopurine in a patient receiving thiopurine therapy. Biomed Chromatogr 1990;4:47-51.[ISI][Medline] [Order article via Infotrieve]
  24. Lennard L, Singleton HJ. High-performance liquid chromatographic assay of the methyl and nucleotide metabolites of 6-mercaptopurine: quantitation of red blood cell 6-thioguanine nucleotide, 6-thioinosinic acid and 6-methylmercaptopurine metabolites in a single sample. J Chromatogr 1992;583:83-90.[ISI][Medline] [Order article via Infotrieve]
  25. Dervieux T, Boulieu R. Simultaneous determination of 6-thioguanine and methyl 6-mercaptopurine nucleotides of azathioprine in red blood cells by HPLC. Clin Chem 1998;44:551-555.[Abstract/Free Full Text]
  26. Erdmann GR, Steury JC, Carleton BC, Stafford RJ, Bostrom BC, Canafax DM. Reversed-phase high-performance liquid chromatographic approach to determine total lymphocyte concentrations of 6-thioguanine, methylmercaptopurine and methylthioguanine in humans. J Chromatogr 1991;571:149-156.[ISI][Medline] [Order article via Infotrieve]
  27. Zimm S, Strong JM. A clinically useful ion-pairing high-performance liquid chromatographic assay for the monophosphate metabolites of thioguanine and mercaptopurine in human neoplastic cells. Anal Biochem 1987;160:1-6.[ISI][Medline] [Order article via Infotrieve]
  28. Breter HJ, Zahn RK. The quantitative determination of metabolites of 6-mercaptopurine in biological materials. II. Advantages of a variable-wavelength HPLC spectrophotometric detector for the determination of 6-thiopurines. J Chromatogr 1977;137:61-68.[ISI][Medline] [Order article via Infotrieve]
  29. Dervieux T, Boulieu R. Identification of 6-methylmercaptopurine derivative formed during acid hydrolysis of thiopurine nucleotides in erythrocytes, using liquid chromatography-mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance assay. Clin Chem 1998;44:2511-2515.[Abstract/Free Full Text]
  30. Su Y, Hon YY, Chu Y, Van de Poll ME. Relling MV. Assay of 6-mercaptopurine and its metabolites in patient plasma by high-performance liquid chromatography with diode-array detection. J Chromatogr B Biomed Sci Appl 1999;732:459-468.[Medline] [Order article via Infotrieve]
  31. McLeod HL, Relling MV, Liu Q, Pui C-H, Evans WE. Polymorphic thiopurine methyltransferase in erythrocytes is indicative of activity in leukemic blasts from children with acute lymphoblastic leukemia. Blood 1995;85:1897-1902.[Abstract/Free Full Text]
  32. Hampton A, Nomura A. Inosine 5'-phosphate dehydrogenase. Site of inhibition by guanosine 5'-phosphate and of inactivation by 6-chloro- and 6-mercaptopurine ribonucleoside 5'-phosphates. Biochemistry 1967;6:679-689.[Medline] [Order article via Infotrieve]
  33. Hampton A. Reaction of ribonucleotides derivatives of purine analogues at the catalytic site of inosine 5'-phosphate dehydrogenase. J Biol Chem 1963;238:3068-3074.[Free Full Text]



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