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 more hydrophobic analytes elute last.

Studying the peak characteristic to each analyte in the HPLC chromatogram helps identify their concentrations within the sample. In this protocol, we employ an RP-HPLC technique to quantify extracellular purine metabolism in chronic lymphocytic leukemic cells.

- To test AMP consumption and adenosine and inosine generation, resuspend 2 million CD19 positive, CD5 positive CLL cells in 250 microliters of serum free medium, and plate the cells in a 48 well plate. Add 250 microliters of 400 micromolar AMP, or AMP plus inhibitors, to obtain a final concentration of 200 micromolar AMP. Include a condition in the absence of the substrate to be used as a blank sample.

Incubate from 30 to 60 minutes at 37 degrees Celsius. At the end of the incubation time, collect 500 microliters of the supernatants in cold microcentrifuge tubes. Immediately centrifuge at 17,000 times G at 4 degrees Celsius for five minutes.

Transfer the supernatants in new 1.5 milliliter microcentrifuge tubes and either proceed to sample preparation for the HPLC runs, or immediately store at minus 80 degrees Celsius. Following pretreatment with the inhibitors and incubation with the AMP substrate, filter the supernatants in new 1.5 milliliter microcentrifuge tubes with 0.2 micron syringe filters. Use one milliliter syringes for filtering.

If the HPLC system is provided with an autosampler, prepare the glass vials for HPLC and use the 100 microliter microinserts for small volumes of sample. Transfer at least 100 microliters of sample in the glass vial with a micropipette, or a glass pasteur pipette. Be careful to transfer without bubbles, and close the vial with the screw cap. Samples are now ready for analysis by reversed phase HPLC.

Select the Run Samples button, and choose New Sample Set Method from the file menu. Select the Empty option, and indicate the number of the vial in the auto-sampler, the sample name, and the injection volume of 50 microliters. Select the Equilibration Column method and the Run Sample method described in the text protocol for all the blank and sample runs that follow. Then, inject 50 microliters of the blank of serum free medium, followed by each sample.

To determine the concentration of purines in each sample, quantify the AMP, adenosine, and inosine, by obtaining the peak areas at the retention times described in the text protocol. Select the Process button, followed by the Integrate option. Choose the start and endpoints of each single peak to obtain the area measurement. This is done manually by tracing a line between the start and end points of the peak.

Proceed to calculate the AMP, adenosine, and inosine consumed and/or produced by the chronic lymphocytic leukemia cells, as described in the text protocol.