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

1. Preparation of Standard and Inhibitors Stock Solutions

1. To prepare AMP, weigh out 0.00345 g of AMP and dissolve it in 5 ml of serum-free medium to have a 2 mM standard solution of AMP.
2. To prepare ADO, weigh out 0.00265 g of ADO and dissolve it in 5 ml of serum-free medium to have a 2 mM standard solution of ADO.
3. To prepare INO, weigh out 0.00268 g of INO and dissolve it in 5 ml of serum-free medium to have a 2 mM standard solution of INO.
4. Prepare a 400 µM solution of AMP, ADO and INO by mixing 1 ml of the 2 mM stock solution in 4 ml of serum-free medium (5 ml final volume).
   1. Make 1:4 dilution of each 400 µM standard solution to obtain a 100 µM concentration by pipetting 500 µl of the 400 µM solution in 1,500 µl of serum-free medium (2 ml final volume).
   2. Prepare serial dilutions of each compound in serum-free medium to obtain the following concentrations: 100 µM – 50 µM – 25 µM – 10 µM – 5 µM – 2.5 µM – 1 µM – 0.5 µM – 0.25 µM.
      Note: For example, pipette 1 ml of the 100 µM solution in 1 ml of serum-free medium (1:2 dilution) to obtain 50 µM concentration and proceed with the remaining dilutions.
5. Prepare a 10 mM stock solution of APCP dissolved in PBS; aliquot and stock at – 30 °C.
6. Prepare 10 mM stock solutions of EHNA hydrochloride, dCF and dipyridamole dissolved in dimethyl sulfoxide (DMSO); aliquot and stock at -30 °C.

2. Program the HPLC Method

1. Prepare a 7 mM ammonium acetate buffer by dissolving 0.770 g of ammonium acetate in 2 L of double deionised water (Buffer A). Adjust to pH 3.0 with hydrochloric acid.
2. Prepare a glass bottle containing at least 2 L of ultrapure acetonitrile for HPLC (Buffer B) and connect the guard column and the column to the HPLC.
   Note: The columns and buffers are at RT while in use.
3. Log into the HPLC software and select the "browse project" button. Go to the "file" menu and select "new method" and then "instrument method".
4. Program the equilibration column method as per Table 1. Set a flow rate of 1.00 ml/min and program the UV detector to read at 260 nm. Save the instrument method and select again “new method” from the “file” menu and then “method set”. Choose the instrument method previously saved and save the current method set with the same name.
5. Repeat the step 2.3 to program the run sample method indicated in Table 2. Set a flow rate of 1.00 mL/min and program the UV detector to read at 260 nm. Save the instrument method and repeat the same procedure as described in step 4.4 to save the method set.
6. Select the "run samples" button, choose "new sample set method" from the "file" menu.
7. Select the "empty" option and enter the number of the vial (in the autosampler), the sample name, the injection volume (50 µl). Select the method set saved before for the "method set" column and enter the total run time (min).
   Note: Enter the equilibration column method (Table 1) in the "method set" column before each sample run and enter the total run time (min).

3. Generation of a Standard Calibration Curve for Each Compound

1. Inject 50 µl of blank (specific serum-free medium, which does not contain adenosine deaminase) and of each standard sample following the methods described in Table 1 and Table 2.
   1. Use the equilibration column method (Table 1) prior to each sample run and set the gradient conditions described in Table 2 to obtain adequate peak separation. Refer to Table 3 for reported retention times of AMP, ADO and INO under these HPLC conditions.
   2. Determine the peak area of AMP, ADO and INO at different concentrations choosing the "process" button in the HPLC software and next the "integrate" option. Alternatively select manually the starting and end points of each peak.
   3. Plot the peak areas against the nominal concentration of each standard to obtain the nine-point calibration curve (100, 50, 25, 10, 5, 2.5, 1, 0.5, 0.25 µM).
   4. Calculate the equation of a straight line for AMP, ADO and INO: y = mx + b,
      where x is the µM concentration and y correspond to the peak area.

   Time	Flow rate (ml/min)	%A	%B
   	1.00	100	0
   1.24	1.00	100	0
   6.22	1.00	2	98
   18.65	1.00	2	98

   Table 1: Equilibration column method. Schematic representation of solvent changes for the equilibration of the column. Buffer A: 7 mM ammonium acetate, pH 3.0. Buffer B: acetonitrile.

   Time	Flow rate (ml/min)	%A	%B
   	1.00	0	100
   3.74	1.00	0	100
   13.71	1.00	15	85
   17.00	1.00	100	0
   20.00	1.00	100	0

   Table 2: Run sample method. Schematic representation of solvent changes for HPLC measurement of purine compounds. Buffer A: 7 mM ammonium acetate, pH 3.0. Buffer B: acetonitrile. 

   	Retention Time	λmax
   AMP	8.00 min	260
   INO	11.00 min	260
   ADO	11.90 min	260

   Table 3: Retention times of purine compounds. Typical retention times observed for AMP, ADO and INO. The UV detector is programmed to read at 260 nm. 

   4. Pretreatment with the Inhibitors and Incubation with the Substrate (AMP)

   1. To test AMP consumption, ADO and INO generation without inhibition of CD73, ADA and nucleoside transporters, resuspend the purified 2 x 10⁶ CD19⁺/CD5⁺ CLL cells in 250 µl of serum-free medium and plate cells in a 48 well plate (or in a 1.5 ml microcentrifuge tube).
   2. Add 250 µl of 400 µM AMP or AMP plus inhibitors to obtain a final concentration of 200 µM AMP. Include a condition in the absence of the substrate to be used as blank sample.
   3. Incubate from 30 to 60 min at 37 °C.
   4. At the end of the incubation time, collect 500 µl of the supernatants in cold microcentrifuge tubes (on ice) and immediately centrifuge at 17,000 x g at 4 °C for 5 min.
   5. Transfer the supernatants in new 1.5 ml microcentrifuge tubes and immediately store at -80 °C or proceed to sample preparation for the HPLC runs.

   5. Samples Preparation for HPLC

   1. Filter the supernatants in new 1.5 ml microcentrifuge tubes with the 0.2 µm syringe filters. Use 1 ml syringes for filtering.
   2. If the HPLC system is provided with an autosampler, prepare the glass vials for HPLC and use the 0.1 ml micro-inserts for small volumes of sample.
   3. Transfer at least 100 µl 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.
   4. Samples are now ready for analysis by RP-HPLC.

   6. HPLC Measurements of Purines 

   1. Select the "run samples" button; choose "new sample set method" from the "file" menu.
   2. Select the "empty" option and indicate: the number of the vial (in the autosampler), the sample name, the injection volume (50 µl).
   3. Select the equilibration column method and the run sample method described in Table 1 and Table 2, respectively, for all the blank and sample runs that follow.
      NOTE: Set the equilibration column method (Table 1) before each sample run.
   4. Inject 50 µl of blank (serum-free medium) and of each sample.
   5. Determine the concentration of purines in each sample, quantify the AMP, ADO and INO by obtaining the peak areas at the retention times described in Table 3.
      1. Select the "process" button and next the "integrate" option. Alternatively choose manually the start and end points of each single peak to obtain the area measurement. This is done by tracing a line between the start and end points of the peak.
      2. Determine the µM concentration applying the equation obtained for the standard curve: y = mx + b, where x represents the µM concentration and y is the area of the peak measured from the unknown sample.
         ​NOTE: An example of the ADO calibration standard curve is reported in Figure 1, where: x = (y area – 981.02)/40026
   6. Considering that 2 x 10⁶ cells were resuspended in 0.500 ml of medium, calculate the µmoles of AMP, ADO and INO consumed and/or produced by 10⁶ CLL cells applying the following proportion:
      µmoles : 1,000 ml = x µmoles : 0.250 ml
      NOTE: The µmoles in 1,000 ml (number µmoles in 1 L) of are equivalent to the µM concentration and x are the µmoles of nucleotide or nucleoside produced by 10⁶ cells. Lastly, convert the µmoles into nmoles consumed and/or produced by 10⁶ CLL cells: nmoles = µmoles x 1,000