Development of a novel P-32-postlabeling method for the analysis of 3 '-azido-3 '-deoxythymidine

3'-Azido-3'-deoxythymidine (AZT) was the first anti-retroviral nucleoside a nalog to be used in the treatment and prevention of acquired immunodeficien cy syndrome (AIDS). A novel P-32-postlabeling assay, based upon thymidine k inase (TK) instead of the conventional T-4 polynucleotide kinase, has been developed for the detection of the levels of AZT incorporated into DNA. Aft er enzymatic digestion of DNA to deoxynucleoside 3'-monophosphates, AZT was isolated by ethyl acetate extraction. The ethyl acetate was evaporated and the AZT was postlabeled by 5'-phosphorylation with [gamma-P-32]ATP and TK. AZT was detected at a level of 51.5 +/- 6.3 (mean +/- SD; n = 4) AZT molec ules/10(5) nucleotides following in vitro incorporation of the drug into hi gh-molecular-weight rat-liver DNA. The P-32-postlabeling method was further validated by the detection of AZT in lung and liver DNA from neonatal mice treated with AZT. The levels of AZT in lung and liver DNA were proportiona l to the dose, with the levels in lung DNA being two-fold higher than those for liver DNA. The limit of detection for the assay was 8 AZT molecules/10 (7) nucleotides using 10 mug of DNA. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.