title: Enzymatic assay for measurement of zidovudine triphosphate in peripheral blood mononuclear cells.
abstract: In this report, we describe a new method to measure intracellular zidovudine triphosphate (ZDV-TP) levels in peripheral blood mononuclear cells (PBMCs) from patients treated with ZDV by utilizing inhibition of human immunodeficiency virus type 1 reverse transcriptase activity by ZDV-TP. Intracellular levels of ZDV-TP were determined with our enzymatic assay in PBMCs isolated from the blood of healthy individuals incubated with different concentrations of labeled ZDV and were validated by high-performance liquid chromatography separation and liquid scintillation counting of the radioactive ZDV-TP. These methods gave virtually identical results over a range of ZDV-TP concentrations from 150 to 900 fmol. ZDV-TP recoveries were over 90%, and the limit of quantitation of ZDV-TP by this method was 20 to 50 fmol. To demonstrate the utility of the method, plasma ZDV and intracellular ZDV-TP concentrations were measured at serial time points over 6 h in 12 human immunodeficiency virus-infected volunteers following a single 100- or 500-mg oral dose of ZDV. Systemic oral clearance rates were similar to those in previous studies with adults but were highly variable (range, 0.86 to 2.75 liters/h/kg of body weight). The area under the plasma concentration versus time curve increased significantly (P < 0.0005) with the dose from a median value of 1.2 mg.h/liter at the lower dose to 4.2 mg.h/liter at the higher dose. Median intracellular ZDV-TP levels ranged from 5 to 57 and 42 to 92 fmol/10(6) cells in volunteers administered 100 and 500 mg of ZDV, respectively. Intracellular ZDV-TP levels rose to a plateau value by 2 h and remained consistent to 6 h. Although the higher dose and higher areas under the curve yielded consistently higher intracellular ZDV-TP levels, systemic pharmacokinetics explains only a modest proportion of the variability in cellular pharmacokinetic. The ZDV-TP bioassay should prove useful in further studies of ZDV metabolism in patient-derived PBMCs at the doses of ZDV currently administered.
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
authors.forename: B L
authors.initials: BL
authors.lastname: Robbins
authors.forename: J
authors.initials: J
authors.lastname: Rodman
authors.forename: C
authors.initials: C
authors.lastname: McDonald
authors.forename: R V
authors.initials: RV
authors.lastname: Srinivas
authors.forename: P M
authors.initials: PM
authors.lastname: Flynn
authors.forename: A
authors.initials: A
authors.lastname: Fridland
mesh_terms: D000998:Antiviral Agents / Q000097:blood* / Q000276:immunology / Q000493:pharmacokinetics; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D054306:Dideoxynucleotides; D054303:HIV Reverse Transcriptase; D006679:HIV Seropositivity / Q000097:blood; D015497:HIV-1 / Q000201:enzymology; D006207:Half-Life; D006801:Humans; D009000:Monocytes / Q000737:chemistry*; D011863:Radioimmunoassay; D018894:Reverse Transcriptase Inhibitors; D013601:T-Lymphocytes / Q000737:chemistry; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000097:blood / Q000276:immunology / Q000493:pharmacokinetics
chemical_list: D000998:Antiviral Agents; D054306:Dideoxynucleotides; D018894:Reverse Transcriptase Inhibitors; C081566:4'-azidothymidine 5'-triphosphate; D015215:Zidovudine; D054303:HIV Reverse Transcriptase