Determination of thiopurine methyltransferase activity in isolated human erythrocytes does not reflect Putative in vivo enzyme inhibition by sulfasalazine.
Both erythrocyte isolation and determination of TPMT activity were performed as described previously in detail (9). Sulfasalazine; S-adenosyl-L-methionine (p-toluenesulfonate salt); n,L-dithiothreitol; 4-aninoacetophenone; trichloroacetic acid; dimethyl sulfoxide (DMSO); triethylamine; and Hanks balanced salt solution (HBSS) were supplied by Sigma. 6-Mercaptopurine was obtained from Fluka, and 6-methylmercaptopurine (6-MMP) was from ICN. Acetonitrile (HPLC grade), phosphoric acid, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and sodium hydroxide were purchased from Merck.
TPMT activity was measured in isolated erythrocytes (see below), after lysis with four volumes of ice-cold water, by an established HPLC procedure (9) based on the conversion of 6-mercaptopurine (pH 7.5 and 37 [degrees]C) to 6-MMP with S-adenosyl-L-methionine as the methyl donor. TPMT activity is expressed as nmol of 6-MMP formed per milliliter of packed erythrocytes per hour [nmol x [(mL Ery).sup.-1] x [h.sup-1]. Results were corrected for sample dilution, hematocrit, and incubation time. Calibration and quality-control materials were prepared by adding a 6-MMP stock solution to the erythrocyte lysate, giving enzyme activities between 4 and 40 nmol * [(mL Ery).sup.-1] x [h.sup-1]. In addition, two separate erythrocyte pools obtained from individuals with either high [14.3 nmol x [(mL Ery).sup.-1] x [h.sup.-1]] or intermediate [6.5 nmol x [(mL Ery).sup.-1] x [h.sup.-1]] TPMT activities were included in every incubation batch as precision controls. The assay was linear up to 50 nmol x [(mL Ery).sup.-1] x [h.sup.-1]], and the detection limit was 0.3 nmol x [(mL Ery).sup.-1] x [h.sup.-1]]. The analytical recovery of 6-MMP ranged between 98.3% and 101.8%. The within-day imprecision for pooled human erythrocytes was <5%, and the between-day imprecision was <7.6%. The respective CV for the control samples enriched with 6-MMP were <2.4% for both within- and between-day studies.
Surplus EDTA-blood from routine laboratory analysis was used to prepare four erythrocyte pools with which the experiments were performed. The erythrocyte isolation procedure was performed as follows: after centrifugation at 800g for 10 min, plasma, leukocytes, and the upper layer of the erythrocytes were removed. After the pellet was washed three times with HBSS (8 mL) and centrifuged for 10 min at 8008, 2 mL of red blood cells was resuspended in 2 mL of HBSS, and the hematocrit was determined with an automatic cell counting device (AC.T 5diff; Beckman Coulter).
For the experiments (Fig. 1A), the erythrocyte pool was divided into five 0.5-mL portions, four of which were incubated for 24 h at 37 [degrees]C in the presence of 80, 160, 320, or 640 [micro]mol/L sulfasalazine. For this purpose, four different sulfasalazine stock solutions were prepared in DMSO and added to the erythrocyte portions in 10-[micro]L aliquots. The fifth portion of erythrocytes was used as a control to which only 10 [micro]L of DMSO was added. The sulfasalazine concentrations were chosen in accordance with the known [IC.sub.50] values for this inhibitor, reported with recombinant human TPMT [78 and 104 [micro]mol/L (4,5)]. Plasma sulfasalazine concentrations after an oral dose are somewhat below, but close to, the [IC.sub.50] (2,4,5). After 24 h, each incubated sample was divided into two portions. The first one was processed directly for determination of TPMT activity. The second one was again washed three times with HBSS (0.5 mL), resuspended in a final volume of ~250 [micro]L of HBSS, and after determination of the hematocrit, was processed for analysis of TPMT activity. All samples were run in duplicate, and the experiment was repeated with another three erythrocyte pools on 3 separate days (total n = 4).
To evaluate differences between groups, the Mann-Whitney test and MedCalc[R], Ver. 4.20 (Mariakerke), were performed. P values <0.05 were considered to be statistically significant. Normally distributed data are presented as the mean (SD); nonnormally distributed data are presented as the median (range).
As shown in Fig. 113, the presence of increasing concentrations of sulfasalazine in the reaction mixture containing the lysed erythrocytes preparations A (Fig. 1A) produced a significant, concentration-dependent inhibition of the enzyme activity. In the absence of the inhibitor, mean TPMT (control samples) activity was 10.3 (2.31) nmol x [(mL Ery).sup.-1] x [h.sup.-1]] in the four separate pools. Incubation with 80, 160, 320, and 640 [micro]mol/L sulfasalazine gave TPMT activities of 9.15 (0.66), 7.70 (1.85), 7.05 (0.87), and 5.73 (0.85) nmol x [(mL Ery).sup.-1] x [h.sup.-1]], respectively. Analytical as well as interindividual variability may be responsible for the up to threefold differences in the degree of inhibition found in multiple experiments using the four different erythrocyte pools.
[FIGURE 1 OMITTED]
In the case of the additionally washed erythrocyte samples (preparations B; Fig. 1A), a slight but not significant decrease in the median TPMT activity [micro]<10% of control) was observed at sulfasalazine concentrations up to 320 [micro]mol/L (Fig. 1B). Although an additional decrease (~35%) in the median TPMT activity was found at a sulfasalazine concentration of 640 /[micro]mol/L, this did not reach statistical significance because of the wide variation in the individual results. These findings suggest that during the erythrocyte isolation procedure, the inhibitor is at least partly removed during the wash step. This assumption, which is compatible with the relatively poor solubility of sulfasalazine in water, was further supported by the fact that the sulfasalazine concentration after the wash step (preparation B) was 15-35% lower than in preparation A over the entire sulfasalazine concentration range tested. These data are based on the differences in the areas of the sulfasalazine peaks between preparations A and B, as codetected in our HPLC procedure (retention time of sulfasalazine, 11.7 min) in relation to the internal standard. The incomplete and variable removal of the inhibitor during the washing step of the routinely applied TPMT activity assay precludes both reliable detection of a putative in vivo inhibition of TPMT by sulfasalazine as well as correct determination of the basic individual TPMT phenotype. In a similar experiment with mesalamine (5-aminosalicylic acid) instead of sulfasalazine (data not shown), we found that this is also true for this aminosalicylic drug.
It can be concluded that the determination of TPMT phenotype in isolated human erythrocytes does not reflect putative in vivo enzyme inhibition by aminosalicylates and cannot be used to investigate and/or predict the risk of thiopurine-induced toxicity potentially resulting from concomitant therapy with thiopurine and aminosalicylate drugs. Assessment of 6-TGN concentrations would thus be a better indicator of in vivo drug-drug interaction between these medications. Furthermore, this could be also expected for any potential reversible inhibitor of TPMT in the sample; thus, probably only the effects of modulators that influence the synthesis/ degradation of the enzyme protein can be detected by the assay system.
The skillful technical assistance of Melanie Fischer is gratefully acknowledged.
DOI : 10.1373/clinchem.2003.026096
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Maria Shipkova, [l] * Paul Dieter Niedmann,  Victor W. Armstrong,  Michael Oellerich,  and Eberhard Wieland   Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Germany; Department of Clinical Chemistry, Georg-August-University Gbttingen, Gbttingen, Germany; * address correspondence to this author at: Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Katharinenhospital, Kriegsbergstrasse 60, D-70174 Stuttgart, Germany; fax 49-7112784809, e-mail email@example.com)
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|Title Annotation:||Technical Briefs|
|Author:||Shipkova, Maria; Niedmann, Paul Dieter; Niedmann, Victor W.; Oellerich, Michael; Wieland, Eberhard|
|Date:||Feb 1, 2004|
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