Hypoxanthine and xanthine levels determined by high-performance liquid chromatography in plasma, erythrocyte, and urine samples from healthy subjects: the problem of hypoxanthine level evolution as a function of time.

The levels of hypoxanthine and xanthine are determined in plasma, erythrocyte, and urine samples by a reverse-phase high-performance liquid chromatographic (HPLC) method. The hypoxanthine concentration increases in erythrocyte and plasma samples when whole blood is stored at room temperature between sampling and centrifugation. Furthermore, the hypoxanthine concentration increases in erythrocyte samples when they are kept apart at room temperature before analysis, whereas the plasma hypoxanthine level remains constant. This result proves an endogenous formation of hypoxanthine in erythrocytes with time, at room temperature. These studies show the necessity of rigorous conditions for the collection, transport, and treatment of blood samples. In order to achieve accurate results, the blood must be centrifuged immediately after collection. The erythrocyte and plasma samples must be stored frozen until deproteinization and HPLC analysis. Under these conditions, the concentrations of hypoxanthine and xanthine in plasma are 2.5 +/- 1 and 1.4 +/- 0.7 microM, respectively. In erythrocyte samples, hypoxanthine concentration reaches 8.0 +/- 6.2 microM.