Stretcher B N, Pesce A J, Wermeling J R, Hurtubise P E
Department of Pathology and Laboratory Medicine, University of Cincinnati School of Medicine, Ohio.
Ther Drug Monit. 1990 Sep;12(5):481-9. doi: 10.1097/00007691-199009000-00013.
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.
已开发出一种方法，使用针对齐多夫定（ZDV）的改良商业放射免疫测定法（RIA）来测量外周血白细胞（PBL）内总磷酸化齐多夫定的浓度。齐多夫定5'-单磷酸很容易合成，并用作RIA改良的程序对照。从健康志愿者中分离出PBL，并与ZDV孵育24小时，以进行代谢磷酸化。对活细胞进行计数、洗涤，并用60%甲醇过夜提取。蒸发后，提取物用pH 9.5的Tris缓冲液复溶。由于RIA抗体与磷酸化齐多夫定的交叉反应最小，样品被分成两部分，其中一部分用碱性磷酸酶（AP）处理以释放磷酸基团。然后对每个部分进行齐多夫定测定。通过从处理部分的浓度中减去未用AP处理部分的浓度来确定磷酸化齐多夫定的浓度。从细胞提取物中回收磷酸化齐多夫定的回收率约为90%，重现性可接受（浓度大于或等于0.25 ng/ml时变异系数小于15%）。在0-50 microM细胞外齐多夫定范围内，细胞内浓度（0.1-1.4 pmoles/10(6)细胞）遵循非线性剂量反应关系，具有浓度依赖性饱和。这些结果证明了测定HIV感染患者体内磷酸化齐多夫定浓度的可行性，这是为这些患者建立治疗范围和最佳给药方案的潜在关键步骤。
