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Technical Briefs |
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Université Claude Bernard Lyon 1, Département de Pharmacie Clinique, de Pharmacocinétique, et d’Evaluation du Médicament, 8 avenue Rockefeller, 69373 Lyon Cedex 08, France
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Hôpital Neuro-Cardiologique, Service Pharmaceutique, 59 boulevard Pinel, 69394 Lyon Cedex 03, France
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St. Jude Children’s Research Hospital, 332 N. Lauderlale St., Memphis, TN 38101
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Hôpital Cardiologique, Service de Bronchopneumologie, 59 boulevard Pinel, 69394 Lyon Cedex 03, France
aauthor for correspondence: fax 33-04-72-35-73-31, e-mail roselyne.boulieu{at}chu-lyon.fr
Thiopurine methyltransferase (TPMT) is a cytosolic enzyme that catalyzes the S-methylation of thiopurine drugs, which are used in cancer chemotherapy and as immunosuppressive agents (1). TPMT activity is controlled by a common genetic polymorphism that contributes to interindividual variability in drug response and, consequently, to implications for thiopurine therapeutic efficacy and toxicity (2). Severe myelosuppression has been reported for TPMT-deficient patients treated with standard doses of thiopurines (3)(4)(5), and high TPMT activity has been associated with the rejection of transplanted organs (6). Because of the clinical significance of the TPMT genetic polymorphism, determination of the TPMT phenotype in red blood cells is routinely performed to optimize and individualize thiopurine treatment (5). Variant alleles of the TPMT gene have been characterized and associated with low TPMT activity (7)(8). Recently, Spire-Vayron de la Moureyre et al. (9) reported that genotypic analysis of TPMT allows the correct determination of metabolic capacity for 87% of individuals. A lower correlation was found for individuals with TPMT activity that was close to the antimode value. Thus, phenotypic analysis may be useful and could be performed concomitantly with genotyping tests. TPMT activity has classically been measured using a radiochemical assay (10). Few HPLC methods using nonradiolabeled calibrators with liquid-liquid extraction (11)(12) or solid-phase extraction (13) have been reported. Here, we report a reversed-phase HPLC method that uses a simple and rapid treatment procedure for the determination of TPMT activity in red blood cells.
Blood samples (5 mL) collected into lithium heparin tubes were centrifuged without delay at 1000g for 10 min at 4 °C. Red blood cells were washed according to a previous protocol (13). The supernatant was stored at -80 °C. 6-Mercaptopurine (6-MP; 10 µL; final concentration, 4 mmol/L) was added to 300 µL of red cell lysates containing 100 µL of 0.15 mol/L potassium phosphate (pH 7.3) and preincubated for 3 min at 37 °C. The reaction was started by adding 30 µL of a mixture of S-adenosyl-L-methionine (SAM), toluene sulfonate salt, and dithiothreitol (final concentration of SAM, 25 µmol/L; final concentration of dithiothreitol, 1 mmol/L). The tubes were incubated for 1 h at 37 °C, and the reaction was stopped by heating for 3 min at 120 °C. After cooling, the tubes were centrifuged at 2000g for 15 min at 15 °C, and an aliquot of the supernatant was analyzed by HPLC.
The methyl 6-MP (Me6-MP) formed was measured using a modification of the HPLC procedure described previously (14). Briefly, Me6-MP was analyzed on a Purospher RP 18-e column (Merck) with a linear gradient elution mode with 0.02 mol/L potassium dihydrogen phosphate (pH 3.5) and a mixture of 0.02 mol/L potassium dihydrogen phosphate (pH 3.5) and methanol (50:50 by volume). The methanol gradient was 0–35% over a period of 10 min. The flow rate was 1.2 mL/min, and Me6-MP was detected at 291 nm using a photodiode array detector. All analyses were performed at ambient temperature. Peak identity was confirmed through library matching by comparison of the unknown peaks with reference spectra.
The influence of 6-MP and SAM concentrations on TPMT activity was assessed using eight concentrations of 6-MP (0–7.5 mmol/L) at a constant SAM concentration of 25 µmol/L and eight concentrations of SAM (0–50.0 µmol/L) at a constant 6-MP concentration of 4 mmol/L. One unit of TPMT activity represents the formation of 1 nmol of Me6-MP per hour per milliliter of packed red cells at 37 °C. Results were normalized on the basis of the hematocrit. The apparent Michaelis–Menten constants (Kms) were estimated from six experiments using the Lineweaver–Burk plot.
The chromatogram of the red blood cell lysate from a subject is shown
in Fig. 1A
. 6-MP and Me6-MP were eluted at 6.2 and 13.1 min,
respectively, with a total run time of 25 min. The mean analytical
recoveries were 89.9% and 88.6% at concentrations of 25 and 150
µg/L, respectively. The calibration curve was linear over Me6-MP
concentrations of 7.5–250 µg/L with a correlation coefficient
>0.998. The quantification limit was 7.5 µg/L of packed cells with a
CV <5% for a 300-µL sample volume. Intraassay and interassay CVs
were <5.5% for replicate analyses of the red blood cell lysate
supplemented with Me6-MP at three different concentrations: 10, 50, and
150 µg/L. Moreover, a quality-control lysate from volunteers in which
TPMT activity (mean, 23.3 nmol/h per mL of packed cells; CV =
7.1%) was determined from replicate analyses during 8 months was
included in each run.
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In the conditions used, the formation of Me6-MP was linear with respect
to a lysate volume of 0–300 µL (r2
= 0.997). The influence of the substrate concentration on TPMT activity
is shown in Fig. 1, B and C
. Kms were
227 and 4.9 µmol/L for 6-MP and SAM, respectively, and the maximum
velocities (Vmax) were 28.1 and 24.9
nmol/h per mL of packed cells. These values are similar to those
reported previously using a radiochemical assay
(10)(15). The TPMT activity determined in a
population of Caucasian subjects was 10.4–41.7 nmol/h per mL of packed
cells with a mean value of 28.9 nmol/h per mL of packed cells. These
preliminary data are in close agreement with the results reported
previously in an adult Caucasian European population
(9)(13). One subject (2.4%) had TPMT in the
intermediate range, 40 (97.6%) subjects had high TPMT activity, and no
patient had low or undetectable TPMT activity. Although the number of
subjects was small, our preliminary results were similar to the
gaussian distribution reported recently in a European population
(9)(12). In the method presented, according to
Szumlanski et al. (15), the chelation step was omitted to
reduce the time of analysis and to simplify the assay. Likewise,
allopurinol was not added to the incubation mixture because of the
absence of the enzyme xanthine oxidase in the erythrocytes. The simple
and rapid sample treatment procedure described allows one to
simultaneously stop the enzymatic reaction and obtain a clean extract
that can be analyzed directly by HPLC. This procedure avoids the use of
acid solutions that may induce potential degradation of thiopurine
nucleotides even at ambient temperature (data not shown), and it avoids
the time-consuming extraction step.
In conclusion, we believe the thiopurine methyltransferase assay described is rapid and reliable because of the lack of laborious liquid-liquid or solid-phase extraction. We also believe that this method could be implemented easily in the clinical laboratory for the phenotypic analysis of TPMT in patients scheduled for thiopurine therapy and could help optimize and individualize thiopurine treatment.
References
The following articles in journals at HighWire Press have cited this article:
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  T. Dervieux, G. Meyer, R. Barham, M. Matsutani, M. Barry, R. Boulieu, B. Neri, and E. Seidman 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 Clin. Chem., November 1, 2005; 51(11): 2074 - 2084. [Abstract] [Full Text] [PDF]
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