Quantitation of urinary nucleosides by high-performance liquid chromatography

It is known that some modified, especially methylated, nucleosides originating from RNA degradation are excreted in abnormal levels in the urine of patients with malignant tumours and they have been proposed as tumour markers. Their measurement could provide a non-invasive diagnostic method, be helpful in the identification of different cancers and in the monitoring of therapeutic effects. In this study, we developed and optimized an analytical procedure to isolate and quantify normal and modified ribonucleosides. The extraction of urinary nucleosides was performed by affinity chromatography on a phenylboronic acid column prior to separation. The reversed-phase high-performance liquid chromatography method allowed a complete separation of sixteen urinary ribonucleosides. The recoveries for the different nucleosides ranged from 83 to 100%, except for xanthosine (66%) and pseudouridine (74%). In normal 24 h urine, the mean levels of thirteen nucleosides (in nmol of nucleoside/mumol of creatinine) were found to be as follows: dihydrouridine (6.37), pseudouridine (25.52), cytidine (0.07), uridine (0.21), 1-methyladenosine (2.19), inosine (0.30), guanosine (0.06), xanthosine (0.59), 3-methyluridine (0.11), 1-methylinosine (1.13). 1-methylguanosine (0.74), adenosine (0.21) and 5'-deoxy-5'-methylthioadenosine (0.12). The first results concerning two kinds of tumours, i.e. breast and floor of mouth tumours, showed some abnormal levels of ribonucleosides. Further experiments are now in progress to measure the modified nucleosides in urine of patients with different forms of Cancer.