In Vitro Measurement of Phosphorylated Zidovudine in Peripheral Blood Leucocytes

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Abstract
A method has been developed to measure the concentration of total phosphorylated zidovudine (ZDV) inside peripheral blood leucocytes (PBLs) using a modified commerical radiommunoassay (RIA) specific for ZDV. ZDV 5''-monophosphate was readily synthetized and used as a procedural control for RIA modification. PBLs were isolated from healthy volunteers and incubated with ZDV for 24 h to allow metabolic phosphorylation. Viable cells were counted, washed, and extracted overnight with 60% methanol. After evaporation, the extract was reconstituted in Tris buffer, pH 9.5. Because of minimal split into two fractions, one of which was treated with alkaline phosphatase (AP) to liberate phosphate groups. Each fraction was then assayed for ZDV. Concentrations of phosphorylated ZDV were determined by subtracting the concentration of the non-AP-treated fraction from that of the treated fraction. Recovery of phosphorylated ZDV from cell extracts was approximately 90%, and reproducibility was acceptable (coefficients of variation < 15% for concentrations .gtoreq. 0.25 ng/ml). Intracellular concentrations (0.1-1.4 pmoles/106 cells) followed a nonlinear dose-response relationship over the range 0-50 .mu.M extracellular ZDV, with concentration-dependent saturation. These results demonstrate the feasibility of determining concentrations of phosphorylated ZDV in HIV-infected patients, a potentially key step in establishing a therapeutic range and optimal dosing regimen for these patients.