EVIDENCE OF ALKALINE-PHOSPHATASE INTERFERENCE IN A ZIDOVUDINE RADIOIMMUNOASSAY

Phosphorylated zidovudine (ZDV) concentrations may provide a link betw een drug exposure and clinical efficacy since these would include the active, intracellular form of the drug, ZDV triphosphate. Many groups are investigating the optimal methodology that can be used to accompli sh this goal. The initial purpose of the present studies was to examin e the effect of the inclusion of cell wash steps on the quantitation o f intracellular ZDV. Ten milliliters of whole blood collected from hea lthy volunteers was spiked with increasing ZDV concentrations (0.187, 0.375, 1.87, and 3.75 mu M), allowed to equilibrate at room temperatur e for 1 h, and separated into whole-blood components by a density grad ient procedure. A mononuclear cell pellet was obtained, reconstituted with 2 ml of phosphate-buffered saline (PBS), and split into two aliqu ots, one of which was not washed at all and the other of which was was hed four times with 1 ml of PBS. All samples were analyzed by ZDV radi oimmunoassay (RIA) after a 1:1 dilution with either 1 mg of alkaline p hosphatase (type 1-S; Sigma) per mi or PBS. Parent ZDV was measured in those samples which were not treated with the enzyme, while total ZDV was measured in those samples which were exposed to alkaline phosphat ase (21 degrees C for 1 h). The result of the difference between the t wo samples is total phosphorylated ZDV. During the experiment, evidenc e of alkaline phosphatase interference with the RTA became apparent, c onfusing interpretation of intracellular ZDV concentrations. This evid ence was based on three sets of data. First, wash samples showed incre ases in ZDV concentrations of as great as 0.127 mu M after exposure to alkaline phosphatase, even though on microscopic inspection the wash samples were acellular. Second, the sum of total ZDV recovered from th e four wash samples plus the washed cell pellet was as much as 14-fold greater than the total ZDV measured in the unwashed cell pellet. Theo retically, at least, these two entities should be equal. Finally, cont rol samples of alkaline phosphatase in PBS (0.5 mg/ml) run directly th rough the assay measured false ZDV levels ranging from 0.002 to 0.075 mu M (0.6 to 20 ng/ml). Alkaline phosphatase is frequently used to mea sure phosphorylated anabolites of ZDV in peripheral blood mononuclear cells. These data show that the particular form of alkaline phosphatas e used may interfere with the ZDV RIA and may confuse the interpretati on of phosphorylated anabolite concentrations of ZDV.