Ribonucleotides of Allopurinol and Oxipurinol in Rat Tissues and Their Significance in Purine Metabolism

Abstract

In the early studies on the metabolism of allopurinol in animals and in man [1,2], it was not possible to find any allopurinol or oxipurinol ribonucleotides in acid-soluble extracts of tissues with methods sensitive enough to detect 10 ⁻⁶M concentrations. Similarly, no such ribonucleotides were detectable in the acid-soluble metabolites of allopurinol in Ehrlich Ascites cells [3] or in human fibroblasts [4]. Although allopurinol was a substrate for hypoxanthineguanine phosphoribosyltransferase (HGPRT) in vitro, the kinetic parameters of allopurinol with human red cell HGPRT [5] indicated that the conditions were not favorable for the formation of this nucleotide in vivo (Table 1). Thus, the K_m for allopurinol was 1 mM, whereas a level of 0.01 mM is the highest level attained after therapeutic doses. The binding of oxipurinol to this enzyme was even poorer, K_i = > 10 mM, and the velocity so low that it was not measurable at the 0.125 mM concentration used in the assay [5], nor at 0.5 mM, used subsequently. Moreover, the relatively low K_m values of hypoxanthine, and xanthine, and the increased level of these bases resulting from xanthine oxidase inhibition, increased the probability that hypoxanthine and xanthine would successfully compete with their respective analogues for HGPRT under in vivo conditions. Nevertheless, the work of Fox [6] and of Kelley [7,8] indicated that the nucleotides of allopurinol and oxipurinol might indeed be formed and be responsible for the orotic aciduria and orotidinuria seen in allopurinol-treated patients. In order to attempt the quantification of such nucleotides, the present studies in rats were undertaken with large doses of ¹⁴C-allopurinol of high specific activity. By the extraction of large amounts of tissue, it was possible to isolate, identify, and quantify the radioactive metabolites. A more detailed account of these investigations will be published elsewhere [9].