A Simple Quantitative Assay for Urinary Adenosine Using Column-Switching High-Performance Liquid Chromatography

Abstract

Adenosine is a physiologically active molecule produced locally in many sites of the body to regulate various cell functions. Measurement of levels of the factor in organs and biological fluids provides clues to its role and we reported an accurate quantitative high-performance liquid chromatography method for urinary adenosine requiring no preliminary sample preparation, other than filtration. Analyses were performed isocratically with a reversed-phase and a molecular exclusion columns connected by a column switch. Each sample was analyzed automatically in 35 min. Linearity could be verified up to 1,000 micromol/L (r = 0.999) and recovery of adenosine was 94.6-98.0%. The coefficients of variation (CV) were established to be 0.56-1.32%, intra-assay, and 1.61-4.67%, inter-assay. Based on analyses of healthy individuals at different ages, we are here able to provide age-related values, infants (1.51 +/- 0.71 micromol/mmol creatinine) and children (1.06 +/- 0.36 and 0.83 +/- 0.27 micromol/mmol creatinine; aged 1-5 and 6-10 years), excreting significantly higher amounts of adenosine than adults (0.44 +/- 0.08 micromol/mmol creatinine). We also measured urinary adenosine from patients suffering from metabolic disease or severe respiratory failure and found that unfavorable pathophysiologic conditions are associated with appreciable elevation of adenosine.