Phenotype determination of thiopurine methyltransferase in erythrocytes by HPLC.
Blood samples (5 mL) collected into lithium heparin tubes were centrifuged without delay at 1000g for 10 min at 4 [degrees]C. Red blood cells were washed according to a previous protocol (13). The supernatant was stored at -80 [degrees]C. 6-Mercaptopurine (6-MP; 10 [micro]L; final concentration, 4 mmol/L) was added to 300 [micro]L of red cell lysates containing 100 [micro]L of 0.15 mol/L potassium phosphate (pH 7.3) and preincubated for 3 min at 37 [degrees]C. The reaction was started by adding 30 [micro]L of a mixture of S-adenosyl-L-methionine (SAM), toluene sulfonate salt, and dithiothreitol (final concentration of SAM, 25 [micro]mol/L; final concentration of dithiothreitol, 1 mmol/L). The tubes were incubated for 1 h at 37 [degrees]C, and the reaction was stopped by heating for 3 min at 120 [degrees]C. After cooling, the tubes were centrifuged at 2000g for 15 min at 15 [degrees]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 [micro]mol/L and eight concentrations of SAM (0-50.0 [micro]mol/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 [degrees]C. Results were normalized on the basis of the hematocrit. The apparent Michaelis-Menten constants ([K.sub.m]s) 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 [micro]g/L, respectively. The calibration curve was linear over Me6-MP concentrations of 7.5-250 [micro]g/L with a correlation coefficient >0.998. The quantification limit was 7.5 [micro]g/L of packed cells with a CV <5% for a 300-[micro]L 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 [micro]g/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. In the conditions used, the formation of Me6-MP was linear with respect to a lysate volume of 0-300 [micro]L ([r.sup.2] = 0.997). The influence of the substrate concentration on TPMT activity is shown in Fig. 1, B and C. [K.sub.m]s were 227 and 4.9 [micro]mol/L for 6-MP and SAM, respectively, and the maximum velocities ([V.sub.max]) 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.
[FIGURE 1 OMITTED]
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.
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Roselyne Boulieu, [1,2] * Martine Sauviat,  Thierry Dervieux, [2,3] Michelle Bertocchi,  and Jean-Francois Mornex 
( Universite Claude Bernard Lyon 1, Departement de Pharmacie Clinique, de Pharmacocinetique, et d'Evaluation du Medicament, 8 avenue Rockefeller, 69373 Lyon Cedex 08, France;
 Hopital Neuro-Cardiologique, Service Pharmaceutique, 59 boulevard Pinel, 69394 Lyon Cedex 03, France;
 St. Jude Children's Research Hospital, 332 N. Lauderlale St., Memphis, TN 38101;
 Hopital Cardiologique, Service de Bronchopneumologie, 59 boulevard Pinel, 69394 Lyon Cedex 03, France;
* author for correspondence: fax 33-04-72-3573-31, e-mail firstname.lastname@example.org)
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|Title Annotation:||Technical Briefs|
|Author:||Boulieu, Roselyne; Sauviat, Martine; Dervieux, Thierry; Bertocchi, Michelle; Mornex, Jean-Francois|
|Date:||May 1, 2001|
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