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Clinical Chemistry 49: 260-268, 2003; 10.1373/49.2.260
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(Clinical Chemistry. 2003;49:260-268.)
© 2003 American Association for Clinical Chemistry, Inc.

Differences in Nucleotide Hydrolysis Contribute to the Differences between Erythrocyte 6-Thioguanine Nucleotide Concentrations Determined by Two Widely Used Methods

Maria Shipkovaa, Victor William Armstrong, Eberhard Wieland and Michael Oellerich

Department of Clinical Chemistry, Georg-August-University Göttingen, D-37075 Göttingen, Germany.

aAddress correspondence to this author at: Abteilung Klinische Chemie, Zentrum Innere Medizin, Georg-August-Universität, Robert Koch Strasse 40, D-37075 Göttingen, Germany. Fax 49-551-3912503; e-mail maria.shipkova{at}med.uni-goettingen.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Measurement of 6-thioguanine nucleotide (6-TGN) concentrations in erythrocytes is widely accepted for use in optimization of thiopurine therapy. Various chromatographic methods have been developed for this purpose. In preliminary experiments we observed a considerable difference between 6-TGN concentrations determined with two widely used methods published by Lennard (Lennard L. J Chromatogr 1987;423:169–78) and by Dervieux and Boulieu (Dervieux T, Boulieu R. Clin Chem 1998;44:551–5). We therefore investigated methodologic differences between the two procedures with respect to hydrolysis of 6-TGNs to 6-thioguanine (6-TG) in more detail.

Methods: We analyzed 6-TGNs in erythrocyte preparations (n = 50) from patients on azathioprine therapy by both methods, using the original protocols. In one set of experiments, we replaced the 0.5 mol/L sulfuric acid in the Lennard method with the 1 mol/L perchloric acid used by Dervieux and Boulieu. In a second set of experiments, we investigated the effect of various dithiothreitol (DTT) concentrations on 6-TG recovery with both methods. In a third set of experiments, we determined the effect of hydrolysis time on both protocols.

Results: Direct comparison of both methods showed that 6-TGN concentrations were, on average, 2.6-fold higher in the Dervieux–Boulieu method over the concentration range tested, although the correlation (r = 0.99; P <0.001) was good. Replacement of sulfuric acid by perchloric acid reduced this difference to ~1.4-fold (r = 0.99; P <0.001). Increasing the DTT concentration enhanced 6-TG recovery. The hydrolysis time used in the Lennard method (1 h) was not sufficient to achieve complete hydrolysis.

Conclusions: The difference between 6-TGN concentrations measured by the two methods is attributable, at least in part, to differences in the extent of nucleotide hydrolysis. For optimization of thiopurine therapy, method-dependent therapeutic ranges are necessary, which precludes comparison of results from clinical studies derived with these methods. Efforts must therefore be made to standardize the analytical procedures for the determination of 6-TGN.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Thiopurine drugs [6-mercaptopurine, 6-thioguanine (6-TG),1 and azathioprine] are widely used in both the treatment of acute lymphoblastic leukemia and autoimmune disorders and in immunosuppressive regimens for the prevention of acute rejection after solid organ transplantation. The active metabolites of these drugs, the 6-thioguanine nucleotides (6-TGNs), are considered to exert their therapeutic effect through incorporation into DNA with subsequent disruption of cell replication (1). The metabolism and bioavailability of thiopurines are subject to wide inter- and intraindividual variation (2)(3)(4)(5) as a result of both genetic and environmental factors (6)(7)(8). There is accumulating evidence that individualized therapy is necessary for optimization of the therapeutic response to these agents (6)(7)(8)(9)(10). Because 6-TGN concentrations in erythrocytes have been found to correlate with the extent of 6-TGN incorporation in peripheral blood leukocyte DNA (11)(12), their routine determination as a surrogate marker has been advocated to assess the effect of thiopurine therapy. High concentrations of erythrocyte 6-TGNs have been associated with the risk of myelosuppression (2)(13)(14)(15)(16)(17) and related complications, such as septicemia and consecutive multiple organ failure. On the other hand, low 6-TGN concentrations may lead to suboptimal treatment, as shown in several clinical studies involving patients suffering from acute lymphoblastic leukemia (18) and Crohn disease (19) as well as recipients of kidney (2) or heart (2) transplants.

Chromatographic methods are exclusively used for the determination of erythrocyte 6-TGN concentrations. Since the synthesis of the first thiopurine drug, 6-mercaptopurine, by Elion in 1951 (20), numerous HPLC procedures have been reported. The method developed by Lennard in 1987 (21) has been the most widely used method in those clinical studies that have served as the basis for establishing treatment-related therapeutic ranges for 6-TGN. This method has been routinely used in our laboratory for many years (2)(7). However, the sample preparation procedure for the Lennard assay is relatively laborious and time-consuming and uses the neurotoxic reagent phenylmercuric acetate (PMA) for the extraction. Furthermore, to allow the simultaneous determination of 6-TGNs and 6-methylmercaptopurine in a single sample, the method was modified, primarily through an alteration of the pH for extraction (22). This is a compromise that is accompanied by suboptimal analytical conditions for both analytes. We therefore sought an alternative HPLC method that would be more rapid and easier to perform and that allowed the simultaneous measurement of 6-methylmercaptopurine with comparable analytical reliability. Dervieux and Boulieu (23) have published a method that appeared to meet these requirements. Surprisingly, we found that the 6-TGN concentrations determined with the new method were always considerably higher than those measured with the Lennard (21) method. The presence of such a difference has major implications for the application of this new method to the monitoring of thiopurine therapy because the therapeutic ranges established with the Lennard method are not valid if the Dervieux–Boulieu procedure is used. In addition, the comparison of results from clinical studies derived with these different analytical procedures is impossible. We therefore performed a detailed comparison of the sample preparation of these two methods.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
drugs and reagents
All reagents used were of the highest available purity. 6-TG (2-amino-6-mercaptopurine), 5-bromouracil (5-BU), D,L-dithiothreitol (DTT), PMA, and Hanks’ balanced salt solution (HBSS) were purchased from Sigma; isoamyl alcohol was from J.T. Baker; and toluene, sulfuric acid, hydrochloric acid, phosphoric acid, sodium hydroxide, potassium dihydrogen phosphate, perchloric acid, and methanol (HPLC grade) were from Merck. The 6-TG (0.1 g/L) and 5-BU (1 g/L) stock solutions were prepared in 0.1 mol/L sodium hydroxide and stored at -80 °C. The toluene–isoamyl alcohol–PMA solution used in the Lennard procedure and the DTT solutions were prepared fresh each day.

erythrocyte preparation
Erythrocytes were obtained from ammonium heparinate- or EDTA-anticoagulated whole blood by centrifugation at 1000g for 10 min. Platelet-rich plasma and the buffy coat, together with the upper layer of erythrocytes, were discarded. Two washing steps were performed with HBSS under the same conditions. Cells were finally resuspended in HBSS to yield a hematocrit of ~0.40, and the exact hematocrit and red blood cell count for calculation of 6-TGN concentration were determined with an automatic hematologic cell counting device (AC.T 5diff; Beckman Coulter). The isolated erythrocytes were portioned into 250-µL aliquots and stored at -80 °C until analysis. Erythrocytes obtained from venous blood of healthy volunteers were processed as above and used for preparation of quality-control samples and calibrators.

Only anonymous excess material from blood samples sent to the laboratory for routine analysis was used to perform all experiments, as well as to prepare the in-house controls. Therefore, according to the guidelines of the local ethics committee, Institutional Review Board approval and/or donor written informed consent were not required.

sample preparation
Lennard method (21).
The procedure was performed as originally described by Lennard (21) and included two steps. The first step consisted of simultaneous denaturation of the erythrocyte proteins and hydrolysis (100 °C for 1 h) of the 6-TGN to 6-TG by sulfuric acid (final concentration, 0.5 mol/L) in the presence of 2 mmol/L DTT to protect thiol groups from oxidation. In the second step, the 6-TG was extracted by formation of the phenylmercury adduct in toluene at alkaline pH. Back-extraction of this organic phase with 0.1 mmol/L hydrochloric acid hydrolyzed the adduct and liberated the free thiopurine into the acid layer.

Erythrocytes (0.8 x 109 cells in 200 µL) were added to 800 µL of DTT (3.75 mmol/L) in a 10-mL glass tube with screw cap. After the addition of 500 µL of 1.5 mol/L sulfuric acid, tubes were incubated at 100 °C for 1 h in a heating block. After cooling, 500 µL of 5 mol/L sodium hydroxide was added to each tube, followed by 8 mL of toluene containing 170 mmol/L isoamyl alcohol and 1.3 mmol/L PMA. The tubes were gently mixed for 10 min and then centrifuged at 900g (5 min). A 6-mL portion of the upper toluene layer was transferred to a new tube and back-extracted with 0.2 mL of 0.1 mol/L hydrochloric acid three times (20 s each time) in a multitube vortex-mixer. The tubes were centrifuged as described above, and 80 µL of the lower acid layer was injected onto the chromatographic column. Whereas the original method described by Lennard (21) used external standard mode for quantification, we used DTT as internal standard in our measurements.

Dervieux–Boulieu method (23).
The procedure was performed as published by Dervieux and Boulieu (23) and included sample deproteinization using 1 mol/L perchloric acid in the presence of 60 mmol/L DTT. This was followed by hydrolysis (100 °C for 45 min) of the 6-TGNs in the separated supernatant to release the 6-TG. There was no further extraction or any other pretreatment of the sample before chromatography. In our laboratory, this procedure was conducted with minor modifications, consisting of the use of an internal standard, 5-BU, and a 50% reduction in the reaction volume. Briefly, we mixed 250 µL of erythrocytes with 20 µL of internal standard (5-BU; 314 µmol/L), 20 µL of DTT (1.1 mol/L), and 50 µL of water for 30 s by vortex-mixing in a 1.5-mL polypropylene tube. To this mixture we added 34 µL of perchloric acid (700 mL/L) and vortex-mixed the tube at the highest frequency; the tube was then centrifuged for 15 min at 3000g. The supernatant (~220 µL) was transferred to brown glass tubes with screw caps, which were then heated for 45 min at 100 °C to hydrolyze the thiopurine nucleotides. After cooling, a 80-µL aliquot was injected into the column.

chromatographic conditions
For chromatographic separation of the free 6-TG after both sample preparation procedures described above, we used a modification (7) of the Lennard chromatographic method (21). A Hypersil C18 column [25 cm x 4.6 mm (i.d.); particle size, 5 µm; MZ Analysentechnik] was used as the stationary phase. The mobile phase consisted of solution A [50 mL of methanol and 950 mL of potassium dihydrogen phosphate, pH 3.0 (final concentration, 20 mmol/L), containing 100 mmol/L triethylamine and 0.5 mmol/L DTT] and solution B [230 mL of methanol and 770 mL of potassium dihydrogen phosphate, pH 3.0 (final concentration, 20 mmol/L), containing 100 mmol/L triethylamine and 0.5 mmol/L DTT]. The analytes were eluted at a flow rate of 1.1 mL/min with the following gradient: 0–1 min, 0% B; 1–4 min, 0–20% B; 4–5.5 min, 20–100% B; 5.5–10 min, 100% B; 10–10.5 min, 100–0% B. The column was maintained at 42 °C. The HPLC system consisted of a chromatographic pump (M480), an automatic injector (GINA 50), a diode array detector (UVD 340S), and a computer interface system controller linked to a PC (Dionex-Gynkotek). 6-TGN concentrations were determined by absorbance at 345 nm in the internal standard mode. Under routine conditions, the assay was calibrated using two-point calibration, 0 and 300 pmol/0.8 x 109 erythrocytes. Deviation from full-range historic calibration curves was always <10%.

The DTT used as internal standard for the Lennard procedure was detected at 322 nm, whereas the 5-BU used as internal standard for the Dervieux–Boulieu procedure was detected at 280 nm. Internal standardization was used to achieve better precision because the samples are kept for 45–60 min at 100 °C, which may cause evaporation. Chromeleon software (Dionex), Ver. 6.3, was used for recording and calculating the data. Ultraviolet spectra of known (calibrators) and unknown peaks were visually compared to exclude possible interferents.

The quality-control samples for both methods [in-house drug-free erythrocyte lysates to which 6-TG (100 and 700 pmol/0.8 x 109 erythrocytes) had been added] were analyzed in each run. The allowed deviation from target values was ± 10%. In addition, pooled erythrocytes from patients undergoing azathioprine therapy constituted a further precision control (accepted range mean ± 2 SD). Thioguanine concentrations were expressed as pmol/0.8 x 109 erythrocytes.

assessment of performance characteristics
The detection limit for 6-TG was calculated for both methods on the basis of a signal-to-noise ratio of 3. For this purpose, the baseline noise signal was obtained from a segment of the respective chromatograms that preceded the 6-TG peak. The lower limit of quantification was set at a 6-TG concentration for which an acceptable within-run imprecision (CV<15%; n = 12) could be obtained. The linearity of the methods was established by constructing calibration curves (n = 3) using drug-free erythrocyte lysates to which increasing 6-TG concentrations (25, 30, 100, 500, 1000, 2000, 3000, and 10 000 pmol/0.8 x 109 erythrocytes) were added. Within- and between-run imprecision and extraction efficiency were studied with drug-free erythrocyte lysates to which 6-TG was added to yield final concentrations of 100, 300, and 700 pmol/0.8 x 109 erythrocytes. The extraction efficiencies for the internal standards 5-BU and DTT were investigated at the concentrations used in the hydrolysis mixture (16.8 µmol/L and 2 mmol/L, respectively). The extraction efficiency was calculated by comparing peak areas obtained for the extracted erythrocyte samples containing 6-TG and DTT or 6-TG and 5-BU with peak areas obtained for aqueous solutions containing the same amount of the compound, which were injected directly on the column without extraction after incubation at 100 °C for 60 or 45 min, respectively. Possible chromatographic interference was evaluated by separate analysis of 50 patient specimens free of 6-TG. The analytical recovery for each method was determined by adding known amounts of 6-TG (100, 300, and 700 pmol/0.8 x 109 erythrocytes) to drug-free erythrocyte lysates. The recovery was calculated by comparing the measured concentrations with the expected concentrations.

effect of hydrolysis conditions and duration of hydrolysis on between-method differences measured in 6-tgn concentrations
There are three major differences between the two methods with respect to the hydrolysis step: (a) the type and concentration of the acid used; (b) the concentration of the DTT used to prevent thiol oxidation; and (c) the duration of hydrolysis. To investigate whether these could be responsible for the observed differences in measured values, we performed the following experiments:

We analyzed 6-TGNs in erythrocyte preparations from 50 blood specimens obtained from patients on azathioprine therapy according to the two analytical procedures described above. In the first set of experiments, the sulfuric acid (0.5 mol/L) typically used in the Lennard method (21) was replaced by perchloric acid (1 mol/L), which is used in the Dervieux–Boulieu method (23). The hydrolyzed samples were then processed without further modifications according to Lennard: i.e., extraction of the phenylmercury adduct into toluene at basic pH and back-extraction with hydrochloric acid.

In a second set of experiments, we investigated the effect of several DTT concentrations (2, 5, 10, 25, and 60 mmol/L) on 6-TG recovery with both methods, using pooled erythrocytes from patients on azathioprine therapy.

In a third set of experiments, we carefully investigated the effect of the duration of hydrolysis (15, 30, 45, 60, and 75 min at 100 °C) in both protocols. In addition, we repeated time course studies with the Lennard method using perchloric acid (1 mol/L) instead of sulfuric acid. For these experiments, we used pooled erythrocytes from patients on azathioprine therapy.

statistical methods
Method comparisons were performed using the nonparametric regression procedure of Passing and Bablok (24) as well as the procedure described by Bland and Altman (25). For correlation analysis, we used the Spearman rank correlation test. Passing–Bablok calculations were performed with EVAPAK (Ver. 2.08; Boehringer Mannheim). Bland–Altman comparisons and Spearman rank correlation analyses were performed using MedCalc computer software (MedCalc Software).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Shown in Fig. 1 are representative chromatograms of erythrocyte preparations derived from a patient on azathioprine therapy, which were processed according to either Lennard (Fig. 1A ) or Dervieux and Boulieu (Fig. 1B ). The chromatographic analyses for samples prepared by both preparation protocols were free of interferences, and there was baseline separation between peaks. The performance characteristics of both procedures, as established in our laboratory (Table 1 ), were almost identical to those published by the respective authors in their original publications (21)(23). Our observed extraction efficiency for 6-TG with the Lennard procedure was ~40% compared with the 64% reported by Lennard (21). Linear regression analysis yielded slopes of 0.00047–0.00051, y-intercepts of 0.019–0.047, and correlation coefficients of 0.997–0.999 (range; n = 3) for the Lennard method, and slopes of 0.00331–0.00338, y-intercepts of -0.011 to -0.026), and correlation coefficients of 0.999–1.00 (range; n = 3) for the Dervieux–Boulieu procedure. The extraction efficiencies for both internal standards (DTT and 5-BU) were constant over a hydrolysis time up to 75 min and independent of increasing 6-TG concentrations. In addition, the extraction efficiency for 5-BU was independent of increasing DTT concentrations in the hydrolysis step. The extraction efficiencies for all experiments described here did not vary considerably. The CVs for the signals obtained with the internal standards within different batches were 5.4% ± 0.53% for DTT and 5.9% ± 0.35% for 5-BU.



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  		Figure 1. Representative chromatograms of patients’ erythrocyte samples, pretreated according the methods of Lennard (A) or Dervieux and Boulieu (B) and further processed using the chromatographic conditions described in the Materials and Methods.

The samples contained 204 (A) and 558 pmol 6-TGN/0.8 x 109 erythrocytes (B) as determined by the respective methods. Wvl, wavelength; mAU, milliabsorbance units.


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  		Table 1. Performance characteristics of the Lennard (21) and Dervieux–Boulieu (23) methods for determination of erythrocyte 6-TGN concentrations as established in our laboratory.1

Although both methods were calibrated with the same calibrator and the target values of the quality-control samples (also identical) were met with both procedures, the 6-TGN concentrations in patient samples measured with the Dervieux–Boulieu procedure [median (range), 236.5 (37–5210) pmol/0.8 x 109 erythrocytes; n = 50] were ~2.6-fold higher than the corresponding values obtained with the Lennard procedure [115.0 (30–1599) pmol/0.8 x 109 erythrocytes; n = 50]. Despite this large difference, there was an excellent and highly significant correlation between the two methods [r = 0.99; P <0.001; y = 2.64(2.31–2.88)x - 54.73(-82.77 to -22.62) pmol/0.8 x 109 erythrocytes; 68% median distance = 20.89; Sy|x = 50.38; n = 50]. To improve the graphic resolution, Fig. 2A shows the Passing–Bablok comparison between the two methods after exclusion of the highest value. As can be seen, inclusion of the highest value did not greatly impact the regression result because we used Passing–Bablok regression analysis, which is known to be very robust against outliers (24). A difference plot (Fig. 2B ) revealed that the absolute difference between the two methods increased with the magnitude of the measurement and that this relationship was highly significant (r = 0.96; P <0.001).



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  		Figure 2. Passing–Bablok method comparison (A) and Bland–Altman difference plot (B) for the Lennard and Dervieux–Boulieu methods with the highest value excluded from the Passing–Bablok comparison.

(A), Passing–Bablok method comparison of 6-TGN concentrations measured in 49 erythrocyte samples from patients receiving thiopurine therapy, prepared using the Lennard and the Dervieux–Boulieu pretreatment procedures. Solid line, regression line; dotted line, line of identity. Regression statistics: y = 2.58x - 50.2 pmol/0.8 x 109 erythrocytes; r = 0.98; P <0.001. (B), Bland–Altman blot comparing 6-TGN concentrations measured using the Lennard and Dervieux–Boulieu pretreatment procedures in all 50 erythrocyte samples investigated. r = 0.96; P <0.001. Erys, erythrocytes.

Replacement of the sulfuric acid (0.5 mol/L) originally used for hydrolysis in the Lennard procedure (21) with perchloric acid (1 mol/L) reduced the difference to 1.4-fold [r = 0.99; y = 1.37(1.24–1.53)x - 34.85(-64.95 to -19.05) pmol/0.8 x 109 erythrocytes; 68% median distance = 30.04; Sy|x = 50.88; n = 50]. For the sake of clarity, Fig. 3A shows the Passing–Bablok comparison between the two methods after exclusion of the highest value. Again the Bland–Altman difference plot (Fig. 3B ) revealed that the absolute difference between the two methods increased with the magnitude of the measurement, and the relationship remained significant (r = 0.70; P <0.001).



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  		Figure 3. Passing–Bablok (A) method comparison and Bland–Altman (B) difference plot for the modified Lennard (perchloric acid used in place of sulfuric acid) and the Dervieux–Boulieu method with the highest value excluded from the Passing–Bablok comparison.

(A), Passing–Bablok method comparison of the 6-TGN concentrations measured in 49 erythrocyte preparations from the same patients shown in Fig. 2AUp . Samples were prepared using the modified Lennard (0.5 mol/L sulfuric acid originally used for hydrolysis replaced by 1 mol/L perchloric acid) and the Dervieux–Boulieu pretreatment procedures. Solid line, regression line; dotted line, line of identity. Regression statistics: y = 1.35x - 32.9 pmol/0.8 x 109 erythrocytes; r = 0.98; P <0.001. (B), Bland–Altman plot comparing 6-TGN concentrations measured using the modified Lennard pretreatment procedure and the Dervieux–Boulieu pretreatment procedure in all 50 erythrocyte samples investigated. r = 0.70; P <0.001. Erys, erythrocytes.

Because the two methods use different concentrations of DTT (2 mmol/L for the Lennard procedure and 60 mmol/L for the Dervieux–Boulieu procedure), we investigated the influence of the DTT concentration on the recovery of 6-TG. For the Dervieux–Boulieu method (23), increasing the DTT concentrations from 2 to 60 mmol/L increased the recovery of 6-TG (Fig. 4 ). The results also remained similar when external standard mode was used for quantification (data not shown). However, in the case of the Lennard procedure (21), DTT concentrations >2 mmol/L led to extremely low 6-TG recoveries, presumably because of poor recoveries in the extraction step, which precluded quantification.



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  		Figure 4. Effect of DTT concentration on 6-TG recovery after sample preparation according to Dervieux and Boulieu.

Values are means of duplicate determinations. Erys, erythrocytes.

We next investigated the effect of duration of hydrolysis at 100 °C in both protocols, using times ranging from 15 to 75 min. The method-dependent 6-TG concentration-time curves are presented in Fig. 5 , A and B. A minimum hydrolysis time of 30 min is necessary for complete hydrolysis of the 6-TGN in the Dervieux–Boulieu method (Fig. 5A ). In the original publication, the authors used a hydrolysis time of 45 min. In the Lennard procedure, a continual increase in 6-TG values was observed in the time periods investigated, which was rapid up to a hydrolysis time of ~60 min and slower thereafter. Although the experiment was extended up to 4 h (data not shown), the curve still did not reach a plateau. The time course studies were repeated with the Lennard method using perchloric acid (1 mol/L) instead of sulfuric acid (0.5 mol/L). As shown in Fig. 5C , after this modification, the 6-TG concentration-time curve reached a plateau in a time period comparable to that observed with the Dervieux–Boulieu procedure. The results also remained similar when external standard mode was used for quantification (data not shown).



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  		Figure 5. Effect of the duration of hydrolysis on 6-TG recovery after sample preparation according to the Dervieux–Boulieu (A) and original (B) and modified (C) Lennard procedures.

In the modified Lennard procedure (C), the sulfuric acid (0.5 mol/L) originally used for hydrolysis was replaced by perchloric acid (1 mol/L). Values are means of duplicate determinations. Arrows indicate hydrolysis time used in the original methods. Erys, erythrocytes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Determination of erythrocyte 6-TGN concentrations can assist clinicians in optimizing therapeutic response to therapy with thiopurine drugs. Multiple assays have been established to monitor 6-TGN concentrations. These assays differ in the matrices used, such as isolated erythrocytes (21)(22)(23)(26)(27)(28)(29)(30)(31), whole blood (32), isolated lymphocytes (33)(34), leukocyte DNA (11)(35), and plasma (36)(37)(38)(39). For routine drug monitoring of 6-TGNs, methods based on isolated erythrocytes are predominantly used. Some of these methods measure individual 6-TGN mono-, di-, and triphosphates (27)(29). However, more commonly, free 6-TG is determined after hydrolysis of 6-TGNs at increased temperatures and acidic pH (21)(22)(23)(26)(28)(33). These approaches differ in sample preparation. The Lennard method uses PMA adduct formation in toluene (21)(22) after the acid hydrolysis, whereas the Dervieux–Boulieu method uses only deproteinization (23). Other authors have applied extraction steps using mercurial cellulose resin and 2-mercaptoethanol (29), aluminum ion complexation (37), or ethyl acetate-dichloromethane (33). The extracted samples are subjected to reversed-phase chromatography with either isocratic or gradient elution. The analyte 6-TG is then monitored by ultraviolet detection (21)(22)(23)(26)(29)(36)(37) or fluorescence detection after derivatization with either potassium permanganate (27)(28)(32) or monobromobimane (35). Finally, the results of 6-TGN measurements have been reported in various units, such as pmol/0.8 x 109 erythrocytes (21)(22), nmol/mmol of hemoglobin (40), pmol/100 erythrocytes (31), or pmol/25 mg of hemoglobin (26). This wide variety in technical details and reporting of the results makes it extremely difficult to compare the published data derived from studies. In this report, we have concentrated on two widely used methods that use the same matrix, chromatographic conditions, detection mode, and units. Despite good correlation between the methods, there was a 2.6-fold difference in the measured 6-TGN concentrations in patient samples. These findings illustrate the importance of the sample pretreatment step in 6-TGN determination. The methods were calibrated with the same calibrator, and each method met the target values for the identical quality-control samples. The principal difference between calibrator/quality-control samples and patients samples is that free 6-TG is added to the former samples, whereas in the latter samples the 6-TGNs must first be hydrolyzed to free 6-TG before quantification. We therefore presumed that the differences between hydrolysis protocols of the two methods could be of particular importance for the differences found.

From the results of our experiments, it seems that the procedure of Dervieux and Boulieu (23) produces more complete conversion of erythrocyte 6-TGNs to 6-TG, leading to higher measured 6-TG concentrations. Multiple factors may be responsible for this observation. Of particular importance are the acid used and the DTT concentration. Furthermore, the hydrolysis step must be allowed to reach completion. We presume that the use of perchloric acid produces more complete protein precipitation, leading to faster liberation of the 6-TGNs from the cell lysates to become available for hydrolysis, than does the use of sulfuric acid. Alternatively, it could be also true that perchloric acid reacts more readily and completely with 6-TGNs to form 6-TG than does sulfuric acid. The observation that increasing the DTT concentrations reduced the extraction efficiency for 6-TG in the Lennard method may be attributable to a competition between DTT and 6-TG for adduct formation with PMA.

In support of our results indicating the importance of the hydrolysis step for 6-TGN determination, Lowry et al. (41) have recently reported an ~1.6-fold difference between 6-TGN concentrations measured with a modified version the Lennard method and a modification of an assay published by Erdmann et al. (28), with the values according to Erdmann being lower. However, in line with our investigation, the authors found a high correlation between the results obtained with both methods (41). Lowry et al. (41) did not investigate the reasons for this difference in more detail but suggested a role of hydrolysis time. In fact, the Erdmann procedure uses a hydrolysis time of 45 min, whereas the Lennard method has a hydrolysis time of 60 min (28)(32). In addition, the concentration of sulfuric acid used for the hydrolysis is lower in the Erdmann assay (28)(32). This may further contribute to incomplete hydrolysis, leading to lower measured 6-TG concentrations.

As a consequence of these methodologic differences, the putative therapeutic ranges are method dependent. To date, little attention has been given to this fact. To interpret results properly, method-specific therapeutic ranges should be considered, which are not available for the Dervieux–Boulieu procedure. Efforts should be made to standardize the analytical procedures for the determination of 6-TGNs.


   Acknowledgments
 
We gratefully acknowledge the skillful technical assistance of Tanja Schneider and Melanie Fischer. We thank Dr. E. Schütz for fruitful discussions.


   Footnotes
 
1 Nonstandard abbreviations: 6-TG, 6-thioguanine; 6-TGN, 6-thioguanine nucleotide; PMA, phenylmercuric acetate; 5-BU, 5-bromouracil; DTT, dithiothreitol; and HBSS, Hanks’ balanced salt solution.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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