Extracellular Purine Metabolism: An HPLC-based Assay to Measure Adenosine Generation and Metabolism in Leukemic Cells

- Adenosine, a purine nucleoside present in nominal concentrations in the extracellular space, helps check cell proliferation under physiological conditions. During pathological conditions like leukemia, the extracellular adenosine concentration increases and inhibits immune responses against cancer cells. Adenosine accumulation results either from ATP or ADP nucleotide dephosphorylation, or due to impaired degradation of adenosine to inosine nucleoside.

To measure adenosine generation and consumption, culture cancer cells in the presence of a nucleotide substrate. The adenosine metabolic activity of cells determines the concentrations of various purine molecules present within the culture. Centrifuge to remove the cells and obtain purine molecules in the supernatant.

To separate and quantify purines, transfer the sample into appropriate vials. Load them into a reversed phase, high performance liquid chromatography, or RP-HPLC column, containing a stationary phase immobilized with hydrophobic ligands. While passing through the column, hydrophobic molecules adsorb to the ligands and are retained based on their hydrophobicity. The least hydrophobic molecules flow out first, while mor