The quantitative determination of metabolites of 6-mercaptopurine in biological materials III. The determination of 14C-labeled 6-thiopurines in L5178Y cell extracts using high-pressure liquid cation-exchange chromatography

Abstract

A method is presented for the separation of 6-thiopurine bases and ribonucleosides, of sulphate anions and of common purine bases and oxidized purines by means of high-pressure liquid cation-exchange chromatography using a 0.18 × 100 cm column, filled with Beckman M71 resin, and eluted with 0.4 M ammonium formate, pH 4.6, at a linear flow velocity of 5.2 cm/min at 50°C. The method has been applied to the separation and quantitative determination of 14C-labeled 6-mercaptopurine metabolites in HClO 4 extracts of L5178Y murine lymphoma cells. Distribution patterns of 14C radioactivity within the cells after a 24 h incubation period with (8- 14C)-labeled 6-mercaptopurine have been established. The identification of 6-mercaptopurine metabolites, such as 6-thioxanthosine ribonucleotide, 6-thioinosinic acid, 6-thioguanylic acid, 6-methylthioinosinic acid, and 6-thiouric acid, after the digestion of the extracts with alkaline phosphatase has been confirmed using the behaviour of each compound in enzymatic peak-shifting analyses with purine nucleoside phosphorylase and the corresponding elution volumes of 6-thiopurine bases and ribonucleosides as proofs. According to the specific radioactivity of the (8- 14C)-labeled 6-mercaptopurine batch, the amounts of the various 6-mercaptopurine metabolites in about 6% of the total HClO 4 extract of 1.6 · 10 8 labeled cells have quantitatively been determined as 1–130 pmol. The intracellular concentration of 6-thiopurines was determined at 1.4 · 10 −5 mol/l.