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Clinical Chemistry 51: 2074-2084, 2005. First published September 15, 2005; 10.1373/clinchem.2005.050831
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Right arrow Drug Monitoring and Toxicology
(Clinical Chemistry. 2005;51:2074-2084.)
© 2005 American Association for Clinical Chemistry, Inc.

Drug Monitoring and Toxicology

Liquid Chromatography–Tandem Mass Spectrometry Analysis of Erythrocyte Thiopurine Nucleotides and Effect of Thiopurine Methyltransferase Gene Variants on These Metabolites in Patients Receiving Azathioprine/6-Mercaptopurine Therapy

Thierry Dervieux1,a, Gary Meyer1, Robert Barham1, Mariko Matsutani1, Mary Barry1, Roselyne Boulieu2, Bruce Neri1 and Ernest Seidman3

1 Prometheus Laboratories, San Diego, CA.
2 Department of Clinical Pharmacy, Claude Bernard University, Lyon, France.
3 Division of Gastroenterology, Research Institute of the McGill University Health Sciences Center, Montreal, Quebec, Canada.

aAddress correspondence to this author at: Prometheus Laboratories, 5739 Pacific Center Blvd, San Diego, CA 92121. Fax 858-332-3349; e-mail tdervieux{at}prometheuslabs.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Polymorphic thiopurine S-methyltransferase (TPMT) is a major determinant of thiopurine toxicity.

Methods: We extracted 6-thioguanine nucleotides (6-TGNs) and 6-methylmercaptopurine nucleotides (6-MMPNs) from erythrocytes with perchloric acid and converted them to 6-thioguanine (6-TG) and a 6-methylmercaptopurine (6-MMP) derivative during a 60-min acid hydrolysis step. The liquid chromatography system consisted of a C18 column with an ammonium acetate–formic acid–acetonitrile buffer. 8-Bromoadenine was the internal standard. Analytes were measured with positive ionization and multiple reaction monitoring mode. With PCR–restriction fragment length polymorphism analysis and TaqMan allelic discrimination, common TPMT alleles (*1, *2, *3A, *3B, *3C) were determined in 31 792 individuals. We used perchloric acid extraction, acid hydrolysis, and HPLC with ultraviolet detection to measure erythrocyte 6-TG and 6-MMP nucleotide concentrations in 6189 patients with inflammatory bowel disease receiving azathioprine/6-mercaptopurine therapy.

Results: Intra- and interday imprecision were <10% at low and high analyte concentrations. The conversion of 6-TG and 6-MMP nucleoside mono-, di-, and triphosphates was complete after hydrolysis. Allelic frequency for TPMT variant alleles ranged from 0.0063% (*3B) to 3.61% (*3A). Compared with wild types, TPMT heterozygotes had an 8.3-fold higher risk for 6-TGNs >450 pmol/8 x 108 erythrocytes (concentration associated with increased risk for leukopenia), but an 8.2-fold lower risk for 6-MMPNs >5700 pmol/8 x 108 erythrocytes (concentration associated with increased risk for hepatotoxicity).

Conclusions: The liquid chromatography–tandem mass spectrometry method can be applied to the routine monitoring of thiopurine therapy. The association between TPMT genotype and metabolite concentrations illustrates the utility of pharmacogenetics in the management of patients undergoing treatment with thiopurines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Thiopurines such as 6-mercaptopurine (6-MP)1 and azathioprine are widely used as immunosuppressants in the treatment of inflammatory bowel disease (1).

6-MP is the analog of hypoxanthine, and azathioprine is a 6-MP prodrug. 6-MP is activated to 6-thioinosine monophosphate (6-TIMP) and subsequently to 6-thioguanine (6-TG) nucleotides (6-TGNs) by a multistep process involving several enzymes of the purine salvage pathway (2). This anabolic route is in competition with methylation of 6-TIMP to 6-methylmercaptopurine (6-MMP) nucleotides (6-MMPNs) by polymorphic thiopurine S-methyltransferase (TPMT). This activation is also in competition with 2 catabolic routes (6-MP oxidation to 6-thiouric acid by xanthine oxidase and methylation to 6-MMP by TPMT). The antiproliferative and immunosuppressive effects of thiopurines are associated with incorporation of deoxythioguanine nucleotides into genomic DNA, de novo purine synthesis inhibition by 6-MMPNs(3), and modulation of RAC-1 activity by 6-thioguanosine triphosphate (6-TGTP)(4).

Studies in the late 1970s demonstrated that TPMT activity was polymorphic and transmitted as an autosomal codominant trait (5). Approximately 1 in 300 individuals cannot produce functional TPMT enzyme. Ten percent of the population are heterozygous for this polymorphism and have intermediate concentrations of TPMT activity; the remaining 90% carry 2 wild-type alleles and have full TPMT activity. The molecular basis for TPMT deficiency is well established and consists of 3 nonsynonymous polymorphisms accounting for >90% of the clinically relevant TPMT mutations(6)(7)(8)(9). The resulting amino acid substitutions do not affect the concentrations of TPMT transcript, but render the protein more susceptible to destruction through ubiquitination(10)(11).

6-MP and azathioprine therapy can cause myelosuppression. Because of the extensive shunt of the metabolism toward 6-TGN formation, patients with the homozygous variant TPMT genotype can experience life-threatening myelosuppression after normal doses of thiopurines and can require dosage reductions of up to 10-fold to tolerate therapy (12)(13). More commonly, patient carriers of heterozygous mutations require dosage reductions of 10%–50%(14)(15), but are also at risk of severe myelosuppression(16). In contrast, patients with the wild-type TPMT genotype are less likely to accumulate 6-TGN concentrations in the toxic range but are more likely to accumulate potentially hepatotoxic 6-MMPNs(1)(17)(18).

The value of monitoring of azathioprine therapy with erythrocyte 6-TGN and 6-MMPN concentrations has been established, and therapeutic thresholds associated with increased likelihood efficacy (6-TGNs >235 pmol/8 x 108 erythrocytes), increased risk for leukopenia (6-TGNs >450 pmol/8 x 108 erythrocytes), and increased risk for hepatotoxicity (6-MMPNs >5700 pmol/8 x 108 erythrocytes) can be used to adjust azathioprine dosage to maintain remission and prevent toxicity (1)(18)(19)(20). Chromatographic methods for the determination of intracellular thiopurine nucleotide concentrations in erythrocytes are available(21)(22)(23)(24) and are based on acid hydrolysis of the thiopurine nucleotide moieties to thiopurine bases(22)(23)(24)(25). We report a liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for the quantification of erythrocyte 6-TGNs and 6-MMPNs and describe the contribution of the TPMT gene locus to thiopurine nucleotide concentrations in a large population of patients with inflammatory bowel disease receiving thiopurine therapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
chemicals and preparation of calibrators
6-TG, 6-MMP, 8-bromoadenosine, and dithiothreitol (DTT) were purchased from Sigma Laboratories. Stock solutions were prepared by dissolution in 50 mmol/L sodium hydroxide. 6-Thioguanosine monophosphate (6-TGMP), 6-thioguanosine diphosphate (6-TGDP), 6-TGTP, methyl 6-thioinosine monophosphate (Me6-TIMP), methyl 6-thioinosine diphosphate (Me6-TIDP), and methyl 6-thioinosine triphosphate (Me6-TITP) were purchased from Jena Biosciences. All thiopurine nucleotides were sodium salts with a final concentration of 10 mmol/L, and the purity was >95%. The 4-amino-5-(methylthio)carbonyl-imidazole (6-MMP derivative) was prepared as described previously (26). 8-Bromoadenine (8-BA) was prepared by hydrolysis of 8-bromoadenosine (500 µmol/L) in 1 mol/L HCl for 1 h at 100 °C. HPLC-grade acetonitrile was purchased from Fisher Chemicals, and perchloric acid (700 mL/L) was obtained from Merck.

preparation and treatment of erythrocyte hemolysate
We collected EDTA-whole blood samples from patients who had received azathioprine/6-MP. Samples were prepared as follows: After centrifugation for 5 min at 2000g to separate plasma and buffy coat from erythrocytes, the erythrocytes were washed once with 2 mL of saline and packed by centrifugation for 5 min at 2000g. A 100-µL packed erythrocyte sample was subsequently used to count the number of erythrocytes (Pentra 80 counter; ABX Diagnostics). Packed erythrocytes were stored at –70 °C until analysis. We used the erythrocyte count determined before freezing to normalize metabolite concentrations in micromoles per liter of erythrocytes to pmol/8 x 108 erythrocytes.

We prepared calibration curves by adding known amounts of 6-TG and 6-MMP to a pool of erythrocyte hemolysates isolated from blood bank samples from healthy blood donors. The calibration curves were constructed with 6-TG concentrations of 0.25, 0.50, 1.0, 2.5, 5.0, 10.0, and 20 µmol/L of erythrocytes and 6-MMP concentrations of 2.5, 5.0, 10.0, 25.0, 50.0, 100.0, and 200.0 µmol/L of erythrocytes. Erythrocyte hemolysate (100 µL; ~1.1 x 109 erythrocytes) was homogenized in a 1.5-mL polypropylene tube with 300 µL of water containing 0.2 mol/L DTT and 10 µmol/L 8-BA. We then added 40 µL of 700 mL/L perchloric acid, and the mixture was immediately thoroughly vortex-mixed for 10 s (to precipitate proteins) and centrifuged for 10 min at 12 000g. Acidic supernatant (250 µL) was subsequently transferred into a 12 x 32 mm glass vial containing a 6 x 31 mm glass insert. The vial was capped and heated at 100 °C in a dry block heater (15-mm wells) for 60 min (except during hydrolysis kinetics experiments). After the supernatant had cooled, a 20-µL aliquot was injected directly into the LC/MS/MS system. Three controls were included in each run for 6-TG (final concentrations, 0.5, 2.5, and 10 µmol/L of erythrocytes) and 6-MMP (final concentrations, 5.0, 25.0, and 100.0 µmol/L of erythrocytes).

lc/ms/ms system
The liquid chromatograph was an Agilent 1100 HPLC system consisting of a binary pump, a system controller, and an autoinjector. The chromatographic system was connected to an API 2000 (Applied Biosystems) with an internal diverter (Valco valve). Chromatographic separation was performed on a Waters Atlantis dC18 column (2.1 cm x 150 mm; 3-µm particle size), protected by an Atlantis dC18 10-mm guard column. Mobile phase A consisted of ammonium acetate (2.5 mmol/L) containing 1.0 mL/L formic acid. Mobile phase B consisted of acetonitrile containing 1 mL/L formic acid. Separation of 6-TG and 6-MMP derivative was achieved with an 8-min isocratic elution with 95% mobile phase A–5% mobile phase B at a flow rate of 0.3 mL/min. Eluates were diverted for the first 2.0 min (to avoid contamination with perchloric acid) and introduced into the turbo-ion spray source thereafter. Ionization was achieved in the positive-ion mode with 5500 V ionization. The heater probe was set at 400 °C. Orifice plate potentials consisted of a declustering potential of 10 V, a focusing potential of 400 V, and an entrance potential of 12 V. Sample analysis was performed in the multiple-reaction monitoring mode with the transitions m/z 168->151 for 6-TG, m/z 158->110 for 6-MMP derivative, and m/z 216->199 for 8-BA. Collision energy was set at 40 V with nitrogen as the collision gas. One sample was injected every 8 min. Integration of peak areas and determination of the concentrations were performed with Analyst 1.4 software (Applied Biosystems). Quadratic regression with 1/x weighted concentrations was used.

precision and ion suppression
We determined intra- and interday imprecision by analyzing low, medium, and high concentrations of 6-TG and 6-MMP added to erythrocyte hemolysates. Intraday analyses were performed with 10 enriched replicates, whereas interday imprecision was assessed on 10 different days. We also calculated the percentage difference between the measured concentrations from each enriched sample relative to the target concentration (measured concentration/target concentration x 100%). Imprecision was determined by estimating the CV.

The kinetics of conversion of thiopurine nucleoside monophosphates (6-TGMP and Me6-TIMP), diphosphates (6-TGDP and Me6-TIDP), and triphosphates (6-TGTP and Me6-TITP) to 6-TG and 6-MMP derivatives was determined with nucleotide calibrators added to erythrocyte hemolysate at final concentrations of 10 µmol/L for 6-TGNs and 100 µmol/L for 6-MMPNs.

We assessed the ion suppression (27) caused by the biological matrix by comparing the ion counts after injection of 6-TG, 8-BA, and 6-MMP derivative added to water (final concentrations of 2.5, 10, and 25 µmol/L, respectively) with the ion counts of 6-TG, 8-BA, and 6-MMP derivative (at the same final concentration) added to blank acidic extracts (after acid hydrolysis) from a pool of erythrocytes for 3 consecutive days with 5 replicates each day. Similarly, the ion suppression was assessed at 2 additional final concentrations of 6-TG (1.0 and 10.0 µmol/L) and 6-MMP derivative (10.0 and 100.0 µmol/L) added to a total of 20 different acidic extracts (after acid hydrolysis) prepared from EDTA blood of 20 individuals who were not receiving thiopurine therapy.

method comparison
We compared the LC/MS/MS procedure with the procedure developed by Lennard and Singleton (23) and modified by Cuffari et al.(20) to determine 6-TGN and 6-MMPN concentrations associated with the efficacy and toxicity of thiopurines in patients with inflammatory bowel disease(1). In 1998, the assay was transferred from St. Justine Hospital to Prometheus Laboratories, and minor modifications to the sample treatment procedure were made. A 50-µL erythrocyte hemolysate was homogenized with 500 µL of water, and 500 µL of 3.0 mol/L H2SO4 plus 300 µL of 10 mmol/L DTT were added. The mixture was heated for 1 h (6-TGN) or 5 h (6-MMPN) at 100 °C. After hydrolysis and neutralization with 3.4 mol/L NaOH and 2 mol/L Tris base, 6-TG and 6-MMP derivative were extracted with 0.3 g/L phenylmercuric chloride in methylene chloride. Phenylmercury adducts in the organic layer were subsequently back-extracted with 0.1 mol/L HCL, and the aqueous layer was injected directly into the HPLC system with ultraviolet detection (HPLC-UV). This modified method was compared with the original method in a blinded fashion between St. Justine Hospital and Prometheus Laboratories. Both methods were equivalent for 6-TGN (regression slope = 0.96; coefficient of correlation = 0.98; n = 30) and 6-MMPN (regression slope = 0.85; coefficient of correlation = 0.99; n = 30) concentrations(28). All procedures and method changes were documented in standard operational procedures and approved by the State of New York Laboratory Services.

The method comparison was performed with erythrocyte hemolysates from patients receiving thiopurines and having 6-TGNs and 6-MMPNs measured in our CLIA-certified laboratory. We selected erythrocyte hemolysates with a large dynamic range of 6-TGN and 6-MMPN concentrations. The samples were blinded by a medical technologist, and the metabolite concentrations were measured with the modified phenylmercury adduct extraction method and the LC/MS/MS method. We also compared the LC/MS/MS method to our current in-house method (Dervieux-Boulieu procedure) (24), which uses a similar sample treatment procedure with UV detection.

determination of tpmt genotypes
Common polymorphisms affecting TPMT activity (G238C, G460A, and A719G) were determined from October 2000 to May 2003 with a PCR–restriction fragment length polymorphism (RFLP) analysis procedure (8) and from June 2003 to January 2005 with a real-time TaqMan allelic discrimination method with fluorogenic 3' minor groove binding probes. The primer and probe sequences are presented in Table 1 .


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  		Table 1. Primers and probes sequences for TaqMan allelic discrimination assay.

The TaqMan allelic discrimination method was as follows: genomic DNA was extracted from 200 µL of EDTA whole blood with capture columns according to manufacturer’s instructions (Gentra Systems, Inc) and quantified by a Molecular Devices SpectraMax 250 spectrophotometer (Global Medical Instrumentation, Inc). Genomic DNA was subsequently diluted to 5 ng/µL. The final PCR conditions were 900 nM of each primer and 200 nM of each probe, with 5 ng of genomic DNA and a 1x TaqMan master mixture (Applied Biosystems). PCR conditions consisted of one 2-min cycle at 50 °C followed by a 10-min cycle at 95 °C followed by 45 cycles of 95 °C for 15 s, 58 °C for 15 s, and finally, 60 °C for 45 s. After amplification was complete, endpoint detection of fluorescence was performed at 60 °C. The fluorescence data were analyzed with the ABI prism 7000 allelic discrimination software (Applied Biosystems). For G460A and A176G determinations, each assay contained a negative control (water; no DNA), a heterozygous control, and a homozygous variant control. For G238C determination, each assay contained a negative control and a heterozygous control (no homozygous variant is available at our institution).

Validation of the TaqMan allelic discrimination method consisted of repeatability and reproducibility experiments with representative samples for each polymorphism (wild types and heterozygous and homozygous variants, if available). A correlation study between PCR-RFLP analysis and TaqMan methods was also performed, and concordance was 100% for the 3 polymorphisms (n = 82 samples; data on file at Prometheus Laboratories). All procedures and method changes were documented in standard operational procedures and approved by the State of New York Laboratory Services. A set of proficiency samples established the continuous acceptable performance of our genotyping methods every 6 months.

The TPMT*2 allele contains the 238C polymorphism, the TPMT*3A allele contains both 460A and 719G polymorphisms, the TPMT*3B allele contains the 460A polymorphism, and the TPMT*3C allele contains the 719G polymorphism.

control and patient populations
All TPMT genotypes and erythrocyte 6-TGN and 6-MMPN measurements were performed in our CLIA-certified laboratory by licensed medical technologists (California State). All erythrocyte 6-TGN and 6-MMPN concentrations were measured with a method involving perchloric acid extraction, acid hydrolysis, and HPLC-UV (24). The data were extracted from our database, and all personal information was removed to protect privacy. The data set was maintained with no possibility of identification of personal information. All thiopurine metabolite data were sent directly by gastroenterologists, and all patients presented with inflammatory bowel disease (ulcerative colitis or Crohn disease) diagnosed on the basis of International Classification of Disease, 9th edition (ICD-9) codes provided by the physicians. Total thiopurine nucleotide (6-TGN + 6-MMPN) concentrations had to be >50 pmol/8 x 108 erythrocytes to ensure compliance.

data analysis
Method comparison was performed by unbiased Deming regression (slope, coefficient of correlation, and intercept) with EP Evaluator (Ver. 6) software (David G Rhoads Associates, Inc). Group comparisons were assessed by Kruskal–Wallis ANOVA with Statistica 6.1 software (StatSoft, Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
lc/ms/ms assay of erythrocyte thiopurine nucleotides
A chromatogram of blank erythrocytes vs erythrocytes supplemented with 6-TG and 6-MMP and subjected to the sample treatment procedure is presented in Fig. 1 . Regression correlation coefficients for calibration curves were >0.995 for 6-TG and 6-MMP derivative. The intra- and interday precision data for our method are presented in Table 2 .



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  		Figure 1. LC/MS/MS chromatograms of erythrocyte thiopurines.

(A), erythrocyte control; (B), erythrocyte hemolysate enriched with 6-TG (2.5 µmol/L; m/z 168->151 transition) and 6-MMP (25.0 µmol/L; m/z 158->110). Both the control and hemolysate were subjected to the sample treatment procedure with 8-BA as internal standard (m/z 216->199).


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  		Table 2. Precision data for 6-TG and 6-MMP in erythrocytes.1

The limits of detection, defined as 5 times the signal-to-noise ratio, were 0.10 µmol/L of erythrocytes for 6-TG and 0.50 µmol/L of erythrocytes for the 6-MMP derivative. The limits of quantification were 0.25 µmol/L of erythrocytes for 6-TG and 1.5 µmol/L of erythrocytes for the 6-MMP derivative. This corresponded to quantification limits of 18 pmol/8 x 108 erythrocytes for 6-TG and of 110 pmol/8 x 108 erythrocytes (assuming 1.1 x 109 erythrocytes per 100 µL of packed erythrocytes, the average in our laboratory).

The conversion of 6-TG and 6-MMP nucleoside mono-, di-, and triphosphates to 6-TG and 6-MMP derivative was complete after a 60-min acid hydrolysis at 100 °C (Fig. 2 ). Use of an equimolar mixture of thiopurine nucleotides added to the biological matrix and quantified against 6-TG and 6-MMP calibrators demonstrated complete conversion of 6-TGNs and 6-MMPNs [90%–110% of the expected values and a CV <10% (Table 3 )].



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  		Figure 2. Kinetics of conversion of thiopurine nucleotides during acid hydrolysis.

6-TG and 6-MMP nucleoside mono- (A), di- (B), and triphosphates (C) were added to erythrocyte hemolysates to final concentrations of 10 µmol/L (6-TGNs) and 100 µmol/L (6-MMPNs). Samples were subjected to the deproteinization step with perchloric acid in the presence of DTT. Acid extracts were subsequently heated to convert 6-TGNs to 6-TG (• in each panel) and 6-MMPNs to the 6-MMP derivative ({circ} in each panel). Results are reported as percentage conversion of the thiopurine nucleotide to the base (6-TG or 6-MMP derivative) with the CV (error bars) at each time point (CV of 3 independent experiments 3 different days). Note that the hydrolysis of 6-TGNs [6-TGMP (A), 6-TGDP (B), and 6-TGTP (C)] to 6-TG was faster than that of 6-MMPNs [Me6-TIMP (A), Me6-TIDP (B), and Me6-TITP (C)] to the 6-MMP derivative.


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  		Table 3. Precision data for erythrocyte 6-TGNs and 6-MMPNs after acid hydrolysis.

Ion suppression experiments revealed that the biological matrix affected 6-TG (final concentration, 2.5 µmol/L) ionization by 40.5 (3.9)%, 8-BA (final concentration 2.5 µmol/L) ionization by 8.0 (1.6)%, and 6-MMP derivative (final concentration 25.0 µmol/L) ionization by 8.3 (4.0)% [mean (SD) of 3 experiments on 3 different days with 5 replicates each day from a pool of erythrocyte hemolysates]. In addition, the ion suppression for 6-TG added to 20 different acidic extracts was 43.4% at a final concentration of 1.0 µmol/L [mean (SD) ion counts of 4.07 (0.36) x 103 in acid extracts vs 7.20 (0.36) x 103 in water] and 43.2% at final concentration of 10.0 µmol/L [mean ion count of 4.02 (0.23) x 104 in acid extracts vs 7.08 (0.40) x 104 in water]. Ion suppression was 9.0% for 6-MMP derivative at a final concentration of 10.0 µmol/L [mean ion counts of 5.84 (0.67) x 104 in acid extracts vs 6.42 (0.72) x 104 in water] and 10.1% at a final concentration of 100.0 µmol/L [mean ion counts of 4.87 (0.28) x 105 in acid extracts vs 5.46 (0.48) x105 in water]. Thus, similar ion suppression was observed at various concentrations of analytes and in various acidic extracts (the CV of the ion counts was <15% in the 20 extracts for all concentration tested).

method comparison
In 100 erythrocyte samples from patients undergoing thiopurine therapy, the median 6-TGN concentrations measured with the LC/MS/MS method was 228 pmol/8 x 108 erythrocytes (interquartile range, 156–329 pmol/8 x 108 erythrocytes), and with the HPLC-UV method developed by Lennard and Singleton (23) and modified by Cuffari et al.(20) it was 196 (135–270) pmol/8 x 108 erythrocytes. As shown in Fig. 3 , we observed a 17% difference between the LC/MS/MS method and the phenylmercuric chloride HPLC-UV method (R = 0.98; slope = 1.17; intercept = 3.7 pmol/8 x 108 erythrocytes). Similarly, we observed a difference of 18% between 6-MMPN concentrations determined with the perchloric acid LC/MS/MS method [median (interquartile range) = 3172 (1296–5271) pmol/8 x 108 erythrocytes] compared with the phenylmercuric adduct extraction HPLC-UV method [2504 (1030–4392) pmol/8 x 108 erythrocytes; R = 0.99; slope = 1.18; intercept = 13.2 pmol/8 x 108 erythrocytes]. 6-TGN and 6-MMPN concentrations measured with the LC/MS/MS assay were also similar to 6-TGN and 6-MMPN concentrations measured with the Dervieux-Boulieu method, which uses a similar sample treatment procedure with UV detection [for 6-TGN, median (interquartile range) = 246 (169–319) pmol/8 x 108 erythrocytes; for 6-MMPN, 3174 (1385–5979) pmol/8 x 108 erythrocytes; n = 100]. This difference was evidenced by slopes of 1.012 (R = 0.95; intercept = –12.6 pmol/8 x 108 erythrocytes) for 6-TGNs and 0.988 (R = 0.99; inter-cept = –163 pmol/8 x 108 erythrocytes) for 6-MMPNs (data not shown).



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  		Figure 3. Method comparison.

Erythrocyte 6-TGNs and 6-MMPNs from 100 patients receiving thiopurine therapy were measured by the method developed by Lennard and Singleton (23) and modified by Cuffari et al.(20), and compared with the perchloric acid extraction–acid hydrolysis method with MS/MS detection. (A), 6-TGN concentrations; (B), 6-MMPN concentrations. The slope, intercept, and coefficient correlation (R) are given. Dotted lines represent the y = x regression.

tpmt gene locus: allelic and genotype frequency
From October 2000 to January 2005, a total of 31 742 TPMT genotypes were determined. The allelic frequencies of common TPMT polymorphisms are presented in Table 4 , and the distribution of genotypes is presented in Table 5 . A total of 8.9% of patients carried a variant allele; the TPMT*1/*3A genotype was the most frequent heterozygous TPMT genotype (75%), followed by the TPMT*1/*3C genotype (19%) and the TPMT*1/*2 genotype (6%). A total of 4 patients (0.014%) carried the TPMT*1/*3B genotype. Three of these TPMT*1/*3B genotypes were determined by PCR-RFLP analysis, and 1 was determined by TaqMan allelic discrimination. Given the frequency of the TPMT*1/*3C and TPMT*1/*3B genotypes in this US-based population, we estimated that the frequency for a TPMT*3B/*3C genotype would be 1 in 420 663 individuals (2.4 per 1 million). Carriers of the TPMT homozygous variant represented 0.33% of the population, and 68% of those individuals carried the TPMT*3A/*3A genotype.


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  		Table 4. Allelic frequency in the 31 742 individuals.


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  		Table 5. Distribution of TPMT genotypes in the 31 742 individuals.

tpmt gene locus and thiopurine metabolite concentrations
A total of 6189 patients with inflammatory bowel disease [mean age, 34 (19) years; 52% female] had a metabolite measurement available with the TPMT genotype. The median (interquartile range) 6-TGN concentration was 180 (122–269) pmol/8 x 108 erythrocytes, and the median 6-MMPN concentration was 881 (289–2784) pmol/8 x 108 erythrocytes. Patient carriers of the heterozygous genotype had higher 6-TGN concentrations [400 (253–594) pmol/8 x 108 erythrocytes] than those with the homozygous wild-type genotype [172 (117–244) pmol/8 x 108 erythrocytes; P <0.0001]. In contrast, carriers of the homozygous wild-type genotype had higher 6-MMPN concentrations [1046 (371–3119) pmol/8 x 108 erythrocytes] than those with the heterozygous genotype [104 (below the detection limit to 504) pmol/8 x 108 erythrocytes; P <0.0001]. All patients with the homozygous variant genotype had 6-MMPN concentrations <150 pmol/8 x 108 erythrocytes with 6-TGN concentrations >600 pmol/8 x 108 erythrocytes [median (interquartile range), 2698 (997–5188) pmol/8 x 108 erythrocytes; Fig. 4 ]. There were no significant differences in 6-TGN and 6-MMPN concentrations among heterozygotes (*1/*3A vs *1/3C vs *1/*2; P >0.20; not shown).



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  		Figure 4. Erythrocyte 6-TGN and 6-MMPN concentrations by TPMT genotype.

(A), erythrocyte 6-TGNs and 6-MMPNs were measured in 6189 patients receiving azathioprine or 6-MP therapy. Patient carriers of the heterozygous genotype (590 patients) had higher 6-TGN concentrations than carriers of the homozygous wild-type genotype (5592 patients; P <0.0001). Carriers of the homozygous wild-type genotype had higher 6-MMPN concentrations than carriers of the heterozygous genotype (P <0.0001). Columns, mean; error bars, SE. (B), representative chromatogram of a sample from a patient receiving azathioprine. Erythrocyte 6-TGN and 6-MMPN concentrations were 2.0 and 21.4 µmol/L of packed erythrocytes. After normalization by the erythrocyte count (1.1 x 109 erythrocytes per 100 µL, determined before freezing), 6-TGN concentrations were 145 pmol/8 x 108 erythrocytes and 6-MMPN concentrations were 1556 pmol/8 x 108 erythrocytes. m/z transitions for the internal standard are not shown.

A total of 4198 patients (67.8%) had 6-TGN concentrations <235 pmol/8 x 108 erythrocytes, and 645 patients (10.4%) had 6-TGN concentrations >450 pmol/8 x 108 erythrocytes. 6-MMPN concentrations were >5700 pmol/8 x 108 erythrocytes in 783 patients (12.6%).

The distributions of 6-TGN and 6-MMPN concentrations by TPMT genotype are shown in Table 6 . Patient carriers of the heterozygous TPMT genotype had a higher risk for 6-TGN concentrations >450 pmol/8 x 108 erythrocytes than did patients with the wild-type genotype (relative risk, 8.3; P <0.0001). All patients with the homozygous variant genotype had 6-TGN concentrations in the range associated with leukopenia (>450 pmol/8 x 108 erythrocytes). Patient carriers of the wild-type genotype had a higher risk of reaching 6-MMPN concentrations >5700 pmol/8 x 108 erythrocytes than patients with the heterozygous genotype (relative risk, 8.2; 95% confidence interval, 5.0–17.3; P <0.0001).


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  		Table 6. Therapeutic thresholds of erythrocyte 6-TGNs and 6-MMPNs by TPMT genotype.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
LC/MS has been used to identify novel thiopurine metabolite in plasma (29) and thiopurine metabolite structural modifications during sample treatment procedures(26). To our knowledge, this is the first LC/MS/MS method developed to quantify 6-TGNs and 6-MMPNs, the 2 major metabolites of azathioprine and 6-MP present in erythrocytes. The method uses a sample treatment procedure in which erythrocyte proteins are precipitated by perchloric acid in the presence of DTT(24). The antioxidant DTT reduces disulfide cross-links of thiol-containing nucleotides with those of sulfhydryl residues on proteins. The resulting acidic extract provides the protons necessary to heat-hydrolyze the thiopurine nucleotide covalent base–ribose bond with formation of 6-TG from 6-TGNs and of 4-amino-5-(methyl)thiocarbonyl imidazole from 6-MMPN. Both analytes are subsequently detected by LC/MS/MS. Advantages of our LC/MS/MS method are improved specificity and a shorter run time (8 min) compared with our UV detection method(24). We used a reversed-phase modified dC18 column that provided enhanced retention of polar 6-TG. The ion suppression caused by the biological matrix was significant for 6-TG (the first analyte eluted), but our validation data demonstrate that this was not deleterious to the performance of the method.

The major drawback for the accurate quantification of thiopurine nucleotides is the lack of readily available thiopurine nucleotide standards to establish the efficiency of the conversion of thiopurine nucleotides to their respective products during the acid-hydrolysis step. To investigate this point, we had thiopurine and methylthiopurine nucleoside mono-, di-, and triphosphate synthesized. The kinetics of hydrolysis of thiopurine nucleotides added to erythrocytes revealed that complete conversion of 6-thioguanosine mono-, di-, and triphosphate to 6-TG and of methyl 6-thioinosine mono-, di-, and triphosphate to 6-MMP derivative was achieved during a 60-min hydrolysis at 100 °C. This was also evidenced by the fact that for an equimolar mixture of thiopurine nucleotides quantified against 6-TG and 6-MMP calibrators, the measured concentrations were >90% of the expected values. It is important to note that the conversion of 6-TGNs (6-TGMP, 6-TGDP, and 6-TGTP) to 6-TG was slightly faster than the conversion of 6-MMPNs (Me6-TIMP, Me6-TIDP, and Me6-TITP) to the 6-MMP derivative, probably because the latter process requires the additional conversion of newly formed 6-MMP (from 6-MMPNs) into the derivative. Thus, complete conversion of 6-MMP to the derivative during the sample treatment procedure ensures complete hydrolysis of 6-TGNs to 6-TG. We propose that simultaneous measurement of 6-TG and 6-MMP derivative is mandatory for laboratories such as ours that use 6-TG and 6-MMP calibrators because the complete conversion of 6-MMP to its derivative can be considered as the internal control for the conversion of 6-TGNs to 6-TG.

Our institution began performing commercial assays for the quantification of 6-TGNs and 6-MMPNs in 1999, after 6-TGN and 6-MMPN concentrations were found to be associated with efficacy and toxicity of azathioprine in patients with inflammatory bowel disease (1)(20). Our initial sample treatment procedure used a modification of the method developed by Lennard and Singleton(23) and adapted by Cuffari et al.(20) to establish azathioprine therapeutic thresholds associated with efficacy (6-TGNs >235 pmol/8 x 108 erythrocytes), leukopenia (6-TGNs >450 pmol/8 x 108 erythrocytes), and hepatotoxicity (6-MMPNs >5700 pmol/8 x 108 cells)(18). The method used 2 separate sample treatment procedures and 2 separate chromatographic conditions for the measurement of 6-TGN and 6-MMPN concentrations, respectively. To improve throughput and cost-effectiveness and avoid generation of toxic mercury waste, the sample treatment procedure was changed to the method of Dervieux and Boulieu(24), which quantifies 6-TGNs and 6-MMPNs in a single run. In June 1999, we established equivalency between both analytical procedures (data in file at Prometheus Laboratories). However, because other laboratories have recently found 1.4-fold(30) to 2.6-fold(31) differences in 6-TGN concentrations determined with the phenylmercury adduct extraction vs the perchloric acid extraction methods, we reinvestigated the equivalency of the 2 sample treatment procedures. Our results confirm our previous observations, and we found a difference of 18% between the 2 procedures for 6-TGN and 6-MMPN concentrations in erythrocytes from patients being treated with azathioprine/6-MP. Thus, the 2 analytical methods can be considered equivalent, with an acceptable 18% difference in the thresholds for 6-TGN and 6-MMPN concentrations. As emphasized by Armstrong et al.(32), however, there is a need to standardize the analytical procedures for the determination of thiopurine nucleotides.

The distribution of TPMT genotypes in this large cohort of individuals is consistent with the original distribution reported by Weinshilboum and Sladek (5). Heterozygosity for TPMT occurred at a frequency of 8.9%, and variant homozygosity at a frequency of 0.33%. Among variant TPMT alleles, the TPMT*3A allele was the most common, followed by the TPMT*3C and TPMT*2 alleles. Several studies have established interethnic differences in the frequencies of TPMT alleles, with the TPMT*3A and TPMT*2 alleles most common in Caucasians and the TPMT*3C allele most common in individuals of black or Asian ethnicity(33)(34)(35). In our population, the ethnicities were not known; therefore, the allelic frequencies reported here are representative only of a US-based diverse population. Interestingly, a total of 4 patients had the TPMT*1/*3B genotype, and the frequency of this genotype was extremely low (0.14{per thousand}). We estimated that TPMT*3B/*3C would occur at a frequency of ~2 of 1 million individuals. Because conventional genotyping methods cannot differentiate individual carriers of a TPMT*1/*3A genotype (460G and 719G on the same allele) from carriers of a TPMT*3B/*3C genotype (460G and 719G on the different allele), elegant haplotyping methods were developed to differentiate the TPMT*3B/*3C homozygous variant from the TPMT*1/*3A heterozygous variant(36)(37). However, the low frequency of the TPMT*3B allele in this population and the estimated frequency of the TPMT*3B/*3C genotype raise concerns as to whether haplotyping methods are applicable and cost-effective in the clinical laboratory.

Several studies have demonstrated the cost-effectiveness of screening for TPMT polymorphisms (38)(39), and data from clinical trials have established the value of measuring thiopurine concentrations to optimize therapy(1)(18)(19)(20).

In our study, patients were not enrolled in a controlled clinical trial; therefore, the data must be interpreted cautiously. Our observations in this large cohort, however, implicate the TPMT locus in the accumulation of thiopurine nucleotides. These data also illustrate the dilemma facing physicians. Patients who are carriers of the wild-type genotype are at higher risk for increased hepatotoxic 6-MMPN concentrations and treatment failure than those with the heterozygous genotype (1)(18)(19). Conversely, those with the heterozygous genotype are less likely to experience hepatotoxicity attributable to 6-MMPN formation but are at higher risk for myelosuppression than those with the wild-type genotype. In our study, all patient carriers of the TPMT homozygous variant genotype had 6-TGN concentrations well above the threshold associated with increased risk for leukopenia and were likely overdosed and experiencing toxicity at the time of measurement of 6-TGN concentrations. We emphasize the need to determine TPMT genotype before therapy to avoid potentially severe toxicity attributable to extensive accumulation of 6-TGNs(40)(41)(42). Alternative methods using erythrocyte TPMT phenotyping(43) can also be considered if the patient has not received previous erythrocyte transfusion treatment.


   Acknowledgments
 
We acknowledge our medical director, Dr. Robert Nakamura, and our medical technologists for scrupulous and continuous attention to the quality of results generated in our CLIA-certified laboratory. We also thank Dr. Richard Bogardt for providing his expertise in data management.


   Footnotes
 
1 Nonstandard abbreviations: 6-MP, 6-mercaptopurine; 6-TIMP, 6-thioinosine monophosphate; 6-TG, 6-thioguanine; 6-TGN, 6-thioguanine nucleotide; 6-MMP, 6-methylmercaptopurine; 6-MMPN, 6-methylmercaptopurine nucleotide; TPMT, thiopurine methyltransferase; 6-TGTP, 6-thioguanosine triphosphate; LC/MS/MS, liquid chromatography–tandem mass spectrometry; DTT, dithiothreitol; 6-TGMP, 6-thioguanosine monophosphate; 6-TGDP, 6-thioguanosine diphosphate; Me6-TIMP, methyl 6-thioinosine monophosphate; Me6-TIDP, methyl 6-thioinosine diphosphate; Me6-TITP, methyl-6-thioinosine triphosphate; 8-BA, 8-bromoadenine; UV, ultraviolet; and RFLP, restriction fragment length polymorphism.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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