Modified nucleosides in urine: selective removal and analysis

Abstract

Because excretion of certain nucleosides increases in all types of cancer, detection and treatment of the disease would be enhanced by rapid, simple quantitation of such nucleosides. To facilitate these analyses in urine, we have prepared a specific adsorbent for nucleosides. The affinity adsorbent gel contains an immobilized phenylboronic acid group that specifically binds cis diols, as in ribonucleosides, which can then be quantitatively recovered by elution with acetic acid. The advantage of this boronate gel over other preparations lies in its high capacity in the swollen state (0.26 mmol/ml), so that only small volumes (less than 1 ml) of gel are required for assays. Nucleosides containing a free diol group on the ribosyl group are retained at pH 8.8 in 0.25 mol/liter salt solution with a retention volume of 15 or larger, as compared with values of 1 to 2 for the free bases. Negatively charged species (not including the boronate complex) are eluted earlier than neutral or cationic species. Added nucleosides (neutral, cationic, or anionic) are quantitatively recovered. Because pseudouridine is eluted in a unique position, this property has been used to measure it in urine. The procedures used are chemically mild. For example, we have confirmed the observation of Fink and Adams [Arch. Biochem. Biophys. 126, 27 (1968)] that urine contains N1 methyladenosine and not N6 methyladenosine, the alkaline rearranged product.