Application of proton NMR spectroscopy to measurement of whole-body RNA degradation rates: Effects of surgical stress in human patients

The urinary catabolites, N-2,N-2-dimethylguanosine (DMG), pseudouridine (PSU) and 7-methylguanine (m(7)-Gua) are formed from post-transcriptional methylation of RNA bases and are not reincorporated into RNA upon its degradation. Their quantitative urinary excretion may be used to determine rates of whole body degradation of individual RNA species since DMG occurs exclusively in tRNA, PSU occurs in rRNA and tRNA and m(7)-Gua occurs in all RNA species. Conventional HPLC analysis has several drawbacks since pre-analytical steps may involve selective losses and, under certain conditions, other urinary analytes may co-elute. In the present paper, we report analysis of these compounds by high-field H-1-nuclear magnetic resonance (H-1-NMR) spectroscopy. Urinary concentrations of these metabolites were found to be in agreement with previously published HPLC and ELISA determinations. However, NMR analysis required minimal sample preparation (other than lyophilisation and reconstitution) and was capable of the simultaneous determination of other relevant analytes such as creatinine. This technique was therefore applied to urine samples from patients who had undergone surgical stress and insulin-like growth factor-I (IGF-I) therapy. Surgical stress increased the excretion of DMG and m(7)-Gua. Degradation rates for tRNA and mRNA were also higher in surgically stressed subjects when compared with controls but degradation rates of rRNA decreased by approx. 30%. However, injection of IGF-I (40 mu g/kg s.c.) had no significant effect on the excretion of these nucleosides. These data indicated that IGF-I therapy has no marked effects on RNA turnover following trauma. We suggest that this technique can be applied to study of RNA metabolism in any surgical or medical condition. Furthermore, since only 0.6 mi of urine is required, studies in neonates seem to be feasible.