Urinary excretion of 3-methyladenine in humans as a marker of nucleic acid methylation.

Antisera to 3-methyladenine (3-meAde) were obtained using a novel analogue of 3-meAde which was bound covalently to methylated bovine serum albumin and used as an antigen. 3-meAde keyhole limpet haemocyanin (3-meAde-KLH) was detected at high dilutions of the antisera (1 in 10(4), and 3-meAde itself inhibited the recognition. In an enzyme-linked immunosorbent assay (ELISA), at room temperature, 3-meAde was detected at 1 pmol/well; however, in a non-equilibrium assay at 4 degrees C, a considerable enhancement of sensitivity was obtained, and 3-meAde was detected at 70 fmol/well. Due to the presence of other purines, the direct determination of urinary 3-meAde was not possible, and a high-performance liquid chromatography (HPLC) clean-up step, followed by ELISA, has been developed. Human urine samples have been analysed by this method and the results compared to those obtained by a gas chromatography-mass spectrometric (GC-MS) method.