Measurement of adenosine metabolites and metabolism in isolated tissue preparations

Several pitfalls have been encountered in setting up methodology for the study of adenosine metabolism and quantitation of adenosine metabolites in isolated tissue preparations. Quantitation of metabolites by high-performance liquid chromatography (HPLC) methods has shown that: 1. significant absorbtion of purines to some types of thread (used to tie up tissues in tissue baths) occurs; 2. without particular care in the preparation of tissue baths and medium, significant microbiological contamination can occur; 3. stimulation of guinea pig ileum preparations at 0.2 Hz is associated with weight loss of the tissue and release of purine-degrading enzymes into the bathing medium; 4. stability of purine analogs is dependent on the tissue and species. A method for the measurement of the purines uric acid, xanthine, hypoxanthine, inosine, adenosine, adenine nucleotides, and various substituted nucleotides in medium and tissue samples following incubations of rat vas deferens and guinea pig ileum is presented.