A radioenzymatic assay for plasma adenosine

A method for analysis of plasma adenosine which combines the principles of radioisotope dilution and enzymatic catalysis is presented. Plasma from venous heparinized blood containing the adenosine deaminase inhibitor 2'-deoxycoformycin is mixed with a small amount of [3H]adenosine and extracted with perchloric acid. Using highly purified enzyme and [gamma-32P]GTP as the phosphate donor, the neutralized extract then serves as substrate for adenosine kinase, and the AMP product is purified by high-performance liquid chromatography. Adenosine concentrations in plasma are linearly proportional to 32P/3H ratios in the enzymatically synthesized AMP and are calculated from a standard curve. The advantages of the method are: ease of sample preparation; sensitivity of 20 nM in as little as 0.3 ml plasma; 20 samples per day can be analyzed by a single operator. Care must be used when obtaining plasma since cellular contamination will affect results. Using this assay, human plasma adenosine levels are 0.121 +/- 0.054 microM for males and 0.101 +/- 0.067 microM for females.