In vitro measurement of phosphorylated zidovudine in peripheral blood leucocytes

A method has been developed to measure the concentration of total phosphorylated zidovudine (ZDV) inside peripheral blood leucocytes (PBLs) using a modified commercial radioimmunoassay (RIA) specific for ZDV. ZDV 5'-monophosphate was readily synthesized 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 RIA antibody cross reactivity with phosphorylated ZDV, samples were 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 less than 15% for concentrations greater than or equal to 0.25 ng/ml). Intracellular concentrations (0.1-1.4 pmoles/10(6) cells) followed a nonlinear dose-response relationship over the range 0-50 microM 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.