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Letters to the Editor |
Departments of1 Clinical Pharmacology and Pharmacy2 Gastroenterology and Hepatology, VU University Medical Center, Amsterdam, The Netherlands
aAddress correspondence to this author at: Drs. P. de Graaf, PharmD/MSc, VU medical centre, Clinical Pharmacology and Pharmacy, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands, Fax (+)31-(0)20-444 3525, e-mail Peer.deGraaf{at}vumc.nl
To the Editor:
In a report recently published in this journal (1), Reinshagen et al., who investigated 6-thioguanine nucleotide (6-TGN) concentrations in patients with inflammatory bowel disease (IBD), concluded that standard and adapted dosing of azathioprine led to identical 6-TGN concentrations and remission rates and that therapeutic drug monitoring of thiopurine therapy was of no clinical benefit in patients with a wild-type thiopurine S-methyltransferase (TPMT) genotype.
As Reinshagen et al. point out, monitoring thiopurine metabolites is a more or less accepted method for assessing pharmacotherapeutic compliance. The use of monitoring to assess the achievement of therapeutic goals is still a matter of ongoing discussion, however, and the study conducted by Reinshagen et al. provides valuable data to inform this debate.
Unfortunately, critical analytical problems still plague thiopurine research. Important differences exist in analytical procedures and reference values (2), and the stability of the thiopurine metabolites themselves is an issue as well. Few studies on stability at 5–7 days are available, although some results have been reported by Pike et al. (3) and Sauviat et al. (4). Decreases in 6-TGN concentrations of 2%–4% per day at ambient temperature (3) and up to 75% on day 4 and <35% on day 7 for samples stored at 20 °C and 4 °C (4) were reported. These results indicate that the stability of 6-TGN in blood samples is limited, a pivotal concern in the many countries where blood samples must be shipped to central laboratories for measurement of thiopurine metabolites. In these situations thiopurine metabolite concentrations can be expected to decrease during transport and/or sample storage.
We prospectively investigated the stability of 6-TGN and 6-methylmercaptopurine (6-MMP) metabolites in blood samples from IBD patients visiting our hospital. Blood was sampled in lithium-heparin tubes, homogenized, and immediately divided into equal portions for storage at controlled conditions (room temperature at 22 °C or refrigeration at 4 °C). A validated analytical procedure described by Shipkova et al. (2) was used to measure 6-TG and 6-MMP nucleotides. The assay had precision values of 5.7% and 4.9% (within day) and 6.9% and 7.2% (between day) for 6-TGN and 6-MMP, respectively. To report the measured metabolite concentrations, erythrocytes were isolated, washed, and counted in the final suspensions before analysis. Analysis was performed at baseline and on days 1, 4, 5, 6, and 7 after sampling.
We obtained samples from 10 patients. The (pseudo)median 6-TGN concentration at day 7 decreased significantly to 53% during storage at room temperature (V = 0, P = 0.002, 95% CI 48%–70%); under refrigeration, the median 6-TGN concentration at day 7 decreased to 90% (not significant). Inter- and intraday variation in our analytical method seems an explanation for these results. Concerning 6-MMP, (pseudo)median concentrations at day 7 decreased significantly to 55% (V = 0, P = 0.002, 95% CI 40%–69%) and 86% (V = 2, P = 0.011, 95% CI 75%–96%) during storage at 22 °C and 4 °C, respectively (Table 1
; Wilcoxon rank-sum test). In addition, decreases in (pseudo) medians were significantly less for both metabolites from day 4 to day 7 during refrigeration [paired Wilcoxon rank-sum test]. These data have been reported previously (5).
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Pike et al. (3) reported less dramatic decreases in nucleotide concentrations (14%–28% decrease at day 7), but our findings are more or less equivalent to the results reported by Sauviat et al. (4). Differences may be largely attributable to the study designs (i.e., the exact storage conditions) and the various analytical methods used. Higher storage temperatures can result in more substantial decreases in 6-TGN and 6-MMP concentrations in blood samples, as is clearly demonstrated by our work. Despite a high correlation shown between various analytical methods, nucleotide hydrolysis techniques vary considerably, mainly in the type of acids, D,L-dithiotreitol, and hydrolysis time used in the analytical procedure (2). These variations can ultimately lead to incomplete hydrolysis and subsequent lower measured 6-TGN concentrations.
Apart from these analytical issues, we have again demonstrated an essential and clinically relevant decrease in both 6-TGN and 6-MMP concentrations attributable to sample storage/shipping conditions, findings that are of pivotal importance for the use of therapeutic monitoring in (future) multicenter studies and for interpretation of pharmacological data in clinical practice in patients on thiopurine therapy. In addition, exact storage conditions are often not mentioned in published reports; their omission may partly explain the current controversy concerning thiopurine metabolite research.
Acknowledgments
Grant/funding Support: None declared.
Financial Disclosure: None declared.
References
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