QUANTITATIVE-DETERMINATION OF METABOLITES OF 6-MERCAPTOPURINE IN BIOLOGICAL-MATERIALS .5. QUANTITATION OF INTRACELLULAR METABOLITES OF [MERCAPTOPURINE-S-35-6 IN L5178Y CELLS GROWN IN TIME-COURSE INCUBATES

Abstract

6-Mercaptopurine (6MP) [an antitumor agent] metabolism was quantitatively determined in L5178Y murine lymphoma. Cells grown in time-course incubates with [35S]-6MP were extracted with cold perchloric acid, and the buffered extracts were subjected to high-performance liquid cation-exchange chromatography prior to and after hydrolysis with alkaline phosphatase. Free sulfate, 6-thiouric acid, 6-thioxanthosine, 6-thioguanosine, 6-thioinosine, free 6MP, and 6-methylthioinosine were separated from each other; identified in the radiochromatograms by elution volume, UV spectroscopic data, and enzymatic peak-shifting analyses with purine nucleoside phosphorylase; and quantitatively determined by means of 35S radioactivity. Gross intracellular 35S concentrations remained constant at 5 .times. 10-5 M after 1 h of incubation. 6MP metabolism in L5178Y cells was distinguished into an early phase (to 1 h of incubation) in which 6MP was predominantly catabolized to 6-thiouric acid and free sulfate, into an intermediate phase (to 8 h) in which substantial amounts of free 6MP and of ribonucleotides of 6-thioxanthosine and 6-thioguanosine were present while the concentrations of nonnucleotide oxidation products sharply decreased, and into a late phase (to 24 h) in which the ribonucleotides of 6MP, of 6-thioguanosine and, in particular, of 6-methylthioinosine were the most abundant metabolites.