A sensitive nonisotopic method for the determination of intracellular azidothymidine 5'-mono-, 5'-di-, and 5'-triphosphate.
In order to analyze the efficacy of azidothymidine (AZT), it is important to know intracellular concentrations of AZT metabolites. However, it has been impossible to measure intracellular AZT 5'-monophosphate (AZT-MP), AZT 5'-diphosphate (AZT-DP), and AZT 5'-triphosphate (AZT-TP) without using isotopes. In the present study, we developed a new method to measure intracellular AZT metabolites without radiolabeled compounds. The method employed was a high-performance liquid chromatography (HPLC) system programmed for column switching technique, in which two columns were used: column 1 (TSK-G2000-SW, 300 x 7.5 mm) to preseparate AZT metabolites from major cell components, and column 2 (YMC-A-312-ODS, 150 x 6 mm) to determine the metabolites. The limit of detectability of this system was 3.3 pmol/injection. When MT-4 cells were incubated with various concentrations of AZT, intracellular concentrations of AZT-MP increased in parallel with extracellular AZT. Those of AZT-DP and AZT-TP, however, reached plateaus at 5 and 2 microM of AZT, respectively. In MT-4 and Molt-4 cells incubated with 5 microM AZT, concentrations of AZT-MP increased time dependently, while the AZT-DP/AZT-MP ratios decreased with time. These data suggest that high dose of AZT may not necessarily increase intracellular concentration of AZT-TP. The concentrations of AZT metabolites in peripheral blood mononuclear cells in a patient with AIDS and an asymptomatic carrier were measured; the concentrations were comparable to those in cultured cells. Quantitative analysis of intracellular AZT metabolites without the use of isotopes will increase safety and convenience of measurement, and take an effective step in studying pharmacokinetics of AZT in clinical materials.
Toyoshima
T
T
Department of Infectious Diseases, University of Tokyo, Japan.
Kimura
S
S
Muramatsu
S
S
Takahagi
H
H
Shimada
K
K
D000163:Acquired Immunodeficiency Syndrome / Q000097:blood; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D054306:Dideoxynucleotides; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D008832:Microchemistry; D010766:Phosphorylation; D013942:Thymine Nucleotides / Q000097:blood*; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000097:blood
D054306:Dideoxynucleotides; D013942:Thymine Nucleotides; C064181:3'-azido-3'-deoxythymidine 5'-diphosphate; C057287:3'-azido-3'-deoxythymidine 5'phosphate; D015215:Zidovudine; C052873:zidovudine triphosphate