Supplemental Data
Purine metabolism, recovery, and parallelism were assessed as described in the Figure
Legend.
Supplemental Figure 1. Metabolism and recovery of purines in sputum. A. To assess
metabolism, sputum samples were obtained and processed as described in the methods
both with and without addition of 0.32% sodium citrate (n=4). Samples were then
incubated at 37 ºC for 30 minutes after addition of~50,000 cpm of [3H] ATP, and
concentrations of [3H]-purines analyzed by HPLC as previously described (32). A
significant fraction of the [3H] ATP was metabolized predominantly to [3H] ADP, and
this metabolism could be reduced (though not eliminated) by addition of sodium citrate.
B. To assess purine recovery, equal amounts of [3H] ATP were added to control sputum,
CF sputum, and buffer (n=3 each). The samples were processed as described in Methods,
and total [3H]-purine counts determined as above. Recovery was defined as the
percentage of counts recovered in sputum compared to buffer and was equivalent in both
control and CF sputa. C. To assess parallelism, 1 µM ATP was added to sputum
supernatants from CF subjects and buffer (HBSS) (n=3 each), and the samples were
serially diluted in buffer to 1:10 and 1:100 of the initial concentration. Adenyl purines
were measured by etheno-derivatization and HPLC as described in Methods. The
measured ATP concentrations were strongly correlated with predicted ATP
concentrations in both sputa and buffer samples, indicating that sputum supernatant
matrix did not impact measurement of ATP.