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

Abstract

Abstract 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.